Replication-competent Herpes Simplex Virus Research Articles

Attenuated Replication-Competent Herpes Simplex Virus Expressing an ECM-Modifying Transgene Hyaluronan Synthase 2 of Naked Mole Rat in Oncolytic Gene Therapy.

Herpes simplex virus (HSV) has proven successful in treating human cancer. Since the approval of talimogene laherparepvec (T-VEC) in 2015, HSV has been thoroughly researched to discover novel mechanisms to combat cancer and treat other diseases. Another HSV-based drug, beremagene geperpavec (B-VEC), received approval in 2023 to treat the rare genetic disease dystrophic epidermolysis bullosa, and was also the first clinically approved HSV vector carrying an extracellular matrix (ECM)-modifying transgene. The ECM is a network of macromolecules surrounding cells, which provides support and regulates cell growth and differentiation, the disruption of which is common in cancer. The naked mole rat (NMR) has a thick ECM and a unique mutation in the hyaluronan synthase 2 (HAS2) gene, which has been linked to the high cancer resistance of the species. To study the effect of this mutation in human cancer, we have developed an attenuated, replication-competent HSV vector expressing the NMR-HAS2 gene. The viral replication, transgene expression and cytotoxic effect of the novel vector was studied in glioma cells. Our results show that an attenuated, replication-competent HSV vector expressing a foreign ECM-modifying transgene, namely HAS2, provides an effective tool to study and combat cancer in humans.

A phase 1 trial of RP2, a first-in-class, enhanced potency oncolytic HSV expressing an anti-CTLA-4 antibody as a single agent and combined with nivolumab in patients with advanced solid tumors.

TPS2704 Background: RP2 is a selective replication-competent herpes simplex virus 1 (HSV-1) that contains a codon-optimized sequence for human granulocyte-macrophage colony-stimulating factor (GM-CSF), the gibbon ape leukemia virus surface glycoprotein (GALV-GP) with the R- sequence deleted (R-), GM-CSF, and an anti–cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody-like molecule. These modifications are intended to increase oncolytic potency, cell-to-cell spread, tumor antigen release, and systemic anti-tumor immune activation. RP2 has demonstrated antitumor activity in preclinical models using viruses similar to RP2 expressing murine versions of anti-CTLA-4 and GM-CSF, both alone and in combination with PD1-1 blockade. Methods: This is a Phase 1 multicenter, open-label, study evaluating RP2 ± nivolumab (NCT04336241). Part 1 evaluated escalating doses of monotherapy RP2 with a modified 3 + 3 design. Patients (pts) received intratumoral (IT) injections of up to 10 mL of RP2 into superficial or visceral tumors Q2W x5 across two dose levels (DL1: 105 plaque-forming units (PFU)/mL once, then followed by 106 PFU/mL x4; DL 2: 106 PFU/mL once, then followed by 107 PFU/mL x4). The recommended Phase 2 dose of RP2 was identified as 106 PFU/mL once, followed by subsequent doses of 1 × 107 PFU/mL. In Part 2, up to 60 evaluable pts will be enrolled, including 30 pts in Cohort 2a and 30 in Cohort 2b. Cohort 2a was designed to evaluate the safety and antitumor activity of the combination in pts with solid tumors, and is fully enrolled. Cohort 2b is open for enrollment of pts with advanced or metastatic uveal melanoma, NSCLC, breast cancer, head and neck cancer or GI cancers (NSCLC, breast or GI cancers must have at least one liver tumor suitable for injection). Pts in Part 2 receive intra-tumoral RP2 at the RP2D Q2W for up to 8 doses in combination with 240 mg nivolumab IV Q2W or 480 mg nivolumab IV Q4W for up to two years from the first RP2 dose. In Part 3 (not yet open for enrollment), up to 15 evaluable pts are able to be enrolled to evaluate RP2 monotherapy including at least 10 pts with NSCLC, breast or GI cancers with at least one liver tumor suitable for injection. Reinitiation of up to 8 additional RP2 doses is permitted if prespecified criteria are met. Pts are dosed with RP2 by direct visualization or ultrasound-guided injection into superficial, subcutaneous, or nodal tumors and by image-guided injection into deeper lesions, including visceral lesions. Key inclusion criteria include histologically confirmed advanced or metastatic non-neurological solid tumors, at least one measurable and injectable tumor of diameter ≥1 cm, ECOG ≤1, and adequate hematologic, hepatic, and renal function. Exclusion criteria include prior treatment with oncolytic viruses, acute or chronic hepatitis B or C infection, and HIV infections. Clinical trial information: NCT04336241.

Oncolytic activity of naturally attenuated herpes-simplex virus HF10 against an immunocompetent model of oral carcinoma

Prognosis for advanced oral carcinoma remains poor. Oncolytic virotherapy uses replication-competent viruses to infect and kill only the tumor cells. However, it has been difficult to investigate the oncolytic activity of viruses against oral carcinomas in mouse models. This study established a mouse model of oral cancer and investigated the in vitro and in vivo anti-tumor effects of HF10, a highly attenuated, replication-competent herpes simplex virus (HSV)-1. Mouse tongue cancer was induced by injecting 4-nitroquinoline 1-oxide into the mouse tongue. The murine oral cancer cell line isolated from this tumor, named NMOC1, formed invasive carcinoma within a week when injected into mouse tongue. HF10 successfully infected, replicated, and spread in the cancer cells in vitro. HF10 was able to kill cancer cells isolated from human or mouse tongue tumor. HF10 injection into tongue carcinomas prolonged mouse survival without any side effects or weight loss. Intertumoral injection of GFP-expressing HF10 confirmed that viral spread was confined within the tumors. Immunohistochemical staining showed that HF10 induced infiltration of CD8-positive T cells around HSV-infected cells in the tumor mass, implying increased anti-tumor immunity. We successfully established an oral cancer cell line and showed that HF10 is a promising therapeutic agent for oral cancer.

2298. Infections in Patients Receiving TVEC Therapy

BackgroundOncolytic viral immunotherapy is an emerging cancer treatment, but the infectious complications are not well described outside of clinical trials. Genetically engineered replication competent herpes simplex virus (HSV-1), commercially known as IMLGYIC® (AmGen) or talimogene laherparepvec (TVEC) was the first FDA approved agent in this class and is used for the local intralesional treatment (of unresectable melanoma. TVEC is derived from a wild-type(WT) strain of HSV-1 (JS—1), which is modified to attenuate off-target effects and promote selective proliferation within cancer cells. Despite these changes local and systemic infection with HSV have been reported from trials and is the subject of an FDA mandated post-marketing review. Here we review the infectious complications of the first cohort of patients treated at our institution post-FDA approval.MethodsDemographic and clinical information for 52 adult patients treated for unresectable melanoma with TVEC following FDA approval in 2015 was extracted from the EMR for the period October 1, 2015–June 30, 2018. EMR and microbiologic data were reviewed for evidence of local site reaction and disseminated infection.ResultsNo cases of disseminated HSV infection were identified during the study period. Of cutaneous reactions, none were documented as greater than severity grade 2, based on standard adverse event reporting criteria. 3 (50%) grade 1–2 cutaneous reactions were deemed probable or definitely related to TVEC and described as pruritis or rash. 12 (23%) patients had any microbiologically confirmed infection identified following TVEC therapy; 6 were bacterial (3 UTI, 1BSI, 2 wound). 8 episodes of viral infections occurred (5 respiratory and 3 GI). A single patient was noted to have localized HSV dermal lesions more than one year after the final TVEC.ConclusionNo local or disseminated HSV infections were encountered in the study cohort. Bacterial skin and soft-tissue infection were uncommon. Most other infections were unrelated to TVEC therapy. Real-world review of the use of an HSV-derived oncolytic viral vector therapy mimics reported infectious complications from clinical trials.Disclosures All authors: No reported disclosures.

Engineering HSV-1 Vectors for Gene Therapy.

Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications and with the approval of Glybera (Alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20:1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer, and in malignant melanoma. In fact, Imlygic® (T-VEC, Talimogene laherparepvec, formerly known as OncoVex GM-CSF), displayed efficacy in a recent Phase-III trial when compared to standard GM-CSF treatment alone (Andtbacka et al., J Clin Oncol 31:sLBA9008, 2013), and has since become the first FDA-approved viral gene therapy product used in standard patient care (October 2015) (Pol et al., Oncoimmunology 5:e1115641, 2016). Moreover, increased efficacy was observed when Imlygic® was combined with checkpoint inhibitory antibodies as a frontline therapy for malignant melanoma (Ribas et al., Cell 170:1109-1119.e1110, 2017; Dummer et al., Cancer Immunol Immunother 66:683-695, 2017). In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are completely replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

Efficacy of HER2 retargeted herpes simplex virus as therapy for high-grade glioma in immunocompetent mice

Replication-competent oncolytic herpes simplex viruses (HSVs) are considered a promising therapeutic approach for treatment of high-grade gliomas (HGGs), which are usually resistant to all the available treatments. We previously demonstrated that R-LM113, a recombinant HSV-1 fully retargeted to the human epidermal growth factor receptor 2 (HER2), is safe and prolongs survival of immunodeficient NOD/SCID mice in an intracranial model of HGG. However, because the treatment is designed to be employed on immunocompetent patients, it is necessary to test whether the host immune system impairs the viral efficacy or triggers a potentially fatal reaction. Here we confirmed the safety of R-LM113 in the immunocompetent mouse strain BALB/c, where it does not trigger encephalitis when intracranially inoculated. Then, we set up a syngeneic HGG model expressing HER2 in adult BALB/c mice and evaluated R-LM113 therapeutic efficacy. We found that R-LM113 leads to a significant improvement in animal survival when administered at the time of tumor inoculation, as well as when injected into an already established tumor. This study suggests that the host immune defenses do not curtail the oncolytic antitumor activity of replication-competent HSV R-LM113, which results effective in counteracting tumor growth.

Replication-competent Herpes Simplex Virus Retargeted to HER2 as Therapy for High-grade Glioma

Oncolytic herpes simplex viruses (HSVs) represent a novel frontier against tumors resistant to standard therapies, like glioblastoma (GBM). The oncolytic HSVs that entered clinical trials so far showed encouraging results; however, they are marred by the fact that they are highly attenuated. We engineered HSVs that maintain unimpaired lytic efficacy and specifically target cells that express tumor-specific receptors, thus limiting the cytotoxicity only to cancer cells, and leaving unharmed the neighboring tissues. We report on the safety and efficacy in a high-grade glioma (HGG) model of R-LM113, an HSV recombinant retargeted to human epidermal growth factor receptor 2 (HER2), frequently expressed in GBMs. We demonstrated that R-LM113 is safe in vivo as it does not cause encephalitis when intracranially injected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, extremely sensitive to wild-type HSV. The efficacy of R-LM113 was assessed in a platelet-derived growth factor (PDGF)-induced infiltrative glioma model engineered to express HER2 and transplanted intracranially in adult NOD/SCID mice. Mice injected with HER2-engineered glioma cells infected with R-LM113 showed a doubled survival time compared with mice injected with uninfected cells. A doubling in survival time from the beginning of treatment was obtained also when R-LM113 was administered into already established tumors. These data demonstrate the efficacy of R-LM113 in thwarting tumor growth.

212. Phase I Trial of Intratumoral (IT) Administration of HF10, a Replication-Competent Herpes Simplex Virus Type 1, in Patients with Refractory Superficial Cancer

212. Phase I Trial of Intratumoral (IT) Administration of HF10, a Replication-Competent Herpes Simplex Virus Type 1, in Patients with Refractory Superficial Cancer

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.

Oncolytic herpes simplex virus treatment of metastatic breast cancer

The high prevalence and poor prognosis of breast cancer provides a strong rationale for developing new treatment strategies and preventive and therapeutic agents. Oncolytic replication-competent herpes simplex virus (HSV) can infect tumor cells, replicating and killing the cells by direct cytopathic effect and then spreading within the tumor. Replication of oncolytic HSV leads to the destruction of the infected tumor cell and release of new virions, which are able to infect adjacent cells until potentially all tumor cells are destroyed. In this study, the cytotoxicity of a third-generation oncolytic HSV vector, designated G47Δ, was examined in human breast cancer cell lines, as well as in immortalized and normal breast cells. A pulmonary metastatic model of breast cancer established in Balb/c nude mice was used to evaluate the efficacy of G47Δ treatment. Systemic treatment by intravenous administration of G47Δ for metastatic lung tumors was initiated 14 days after injection of tumor cells. On Day 56, the mice were sacrificed and tumor nodules on the surface of the lung were counted. G47Δ was highly cytotoxic to breast cancer and immortalized breast cells in vitro at low multiplicities of infection (MOI), while normal breast cells remained viable 5 days after infection. In the pulmonary metastatic model, the average number of surface lung tumor nodules in the G47Δ-treated group was approximately 9‑fold less than in the control-treated group. X-gal staining illustrated viral replication and spread in the tumor cells in vitro and in vivo. In conclusion, G47Δ effectively killed human breast cancer cells and immortalized breast cells but not normal breast cells. Systemic administration of G47Δ by tail vein injection was effective in inhibiting the growth of established breast cancer lung metastases.

Strategies for the Rapid Construction of Conditionally-Replicating HSV-1 Vectors Expressing Foreign Genes as Anticancer Therapeutic Agents

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.

Abstract P2-18-01: Treatment of Human Breast Tumors by Oncolytic Herpes Simplex Virus

Abstract Background: Breast cancer is the most common cancer for women in the world, although current therapies have improve the outcome, the breast cancer deaths are still the second highest among cancers in women. Metastasis to the lung frequently results in severe complications, and currently, there are no curative treatment options available for patients with metastetic breast cancer. Oncolytic viral therapy is a new strategy for treating cancers. Replication-competent herpes simplex virus (HSV) can infect, replicate and kill tumor cells by a direct cytopathic effect and then spread within the tumor cells. In this study, the cytotoxicity of HSV vectors, G47Δ were examined on different human breast cancer cell lines, immortalized human breast cell lines, and primary culture normal human breast cells. Pulmonary metastasis model of breast cancer was constructed and the treatment effect of G47D was evaluated. Methods: Human BC cell lines SK-BR-3, MDA-MB-453, and MCF-7, immortalized breast cells MCF-10A and 76N-tert, and cultured primary normal breast cells HMEC1 and HMEC2 were cultured in vitro and infected with G47Δ at different multiplicities of infection (MOIs). The viability of infected cells was measured. G47Δ-infected cells were identified using X-gal histochemistry for LacZ expression. A pulmonary metastatic BC model was established with Balb-c nude mice by tail vein injection of MDA-MB-435 cells. Treatment was initiated 21 days after injection of tumor cells, with the virus group treated twice weekly with four total i.v. administrations of G47Δ (2*107pfu/100ul) and on day 60 mice were sacrificed and tumor nodules on the surface of the lung counted after Indian ink staining. Results: G47Δ was highly cytotoxic to BC cells and 76N-tert in vitro at very low MOI (0.01). For all of these cell lines, more than 85% and 95% cells were killed at MOI=0.01 and MOI=0.1 respectively on the fifth day after infection. However, both HMEC1 and HMEC2 cells were viable even 5 days after infection. A slight effect was found with MCF-10A, of which only 7% and 24.5% were killed at MOI=0.01 and MOI=0.1 respectively on day 5. In the pulmonary metastatic model, the average number of surface lung tumor nodules in the G47Δ group was one, but ten in the control group. X-gal staining illustrated viral replication and spread in the tumor cells in vitro and in vivo. Conclusions: Recently constructed oncolytic HSV vector G47Δ was effective at killing human breast cancer cells and immortalized breast cells, but didn't infect the normal breast cells. The i.v. administrations of G47Δ could effectively cure metastatic breast cancer. Our studies demonstrated that as a novel therapeutic agents, G47Δ was safe and exhibited superior antitumor effects, thereby making it appropriate for human clinical trials. It is worth noting that G47Δ was effective for lung metastatic breast cancer, which could assure the long survival rate of patients with advance breast cancer. The efficiency of G47Δ warrants consideration of clinical application. (Key words) breast cancer; cancer therapy; herpes simplex virus; oncolytic virus; gene therapy; Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-18-01.

Treatment of human breast tumors by oncolytic herpes simplex virus.

e13605 Background: Oncolytic replication-competent herpes simplex virus (HSV) can infect, replicate and kill tumor cells by a direct cytopathic effect and then spread within the tumor. G47Δ is a th...

Imaging of Lymph Node Micrometastases Using an Oncolytic Herpes Virus and [18F]FEAU PET

BackgroundIn patients with melanoma, knowledge of regional lymph node status provides important information on outlook. Since lymph node status can influence treatment, surgery for sentinel lymph node (SLN) biopsy became a standard staging procedure for these patients. Current imaging modalities have a limited sensitivity for detection of micrometastases in lymph nodes and, therefore, there is a need for a better technique that can accurately identify occult SLN metastases.Methodology/Principal FindingsB16-F10 murine melanoma cells were infected with replication-competent herpes simplex virus (HSV) NV1023. The presence of tumor-targeting and reporter-expressing virus was assessed by [18F]-2′-fluoro-2′-deoxy-1-β-D-β-arabinofuranosyl-5-ethyluracil ([18F]FEAU) positron emission tomography (PET) and confirmed by histochemical assays. An animal foot pad model of melanoma lymph node metastasis was established. Mice received intratumoral injections of NV1023, and 48 hours later were imaged after i.v. injection of [18F]FEAU. NV1023 successfully infected and provided high levels of lacZ transgene expression in melanoma cells. Intratumoral injection of NV1023 resulted in viral trafficking to melanoma cells that had metastasized to popliteal and inguinal lymph nodes. Presence of virus-infected tumor cells was successfully imaged with [18F]FEAU-PET, that identified 8 out of 8 tumor-positive nodes. There was no overlap between radioactivity levels (lymph node to surrounding tissue ratio) of tumor-positive and tumor-negative lymph nodes.Conclusion/SignificanceA new approach for imaging SLN metastases using NV1023 and [18F]FEAU-PET was successful in a murine model. Similar studies could be translated to the clinic and improve the staging and management of patients with melanoma.

A Herpes Oncolytic Virus Can Be Delivered Via the Vasculature to Produce Biologic Changes in Human Colorectal Cancer

Genetically engineered herpes simplex viruses (HSVs) can selectively infect and replicate in cancer cells, and are candidates for use as oncolytic therapy. This long-term report of a phase I trial examines vascular administration of HSV as therapy for cancer. Twelve subjects with metastatic colorectal cancer within the liver failing first-line chemotherapy were treated in four cohorts with a single dose (3 x 10(6) to 1 x 10(8) particles) of NV1020, a multimutated, replication-competent HSV. After hepatic arterial administration, subjects were observed for 4 weeks before starting intra-arterial chemotherapy. All patients exhibited progression of disease before HSV injection. During observation, levels of the tumor marker carcinoembryonic antigen (CEA) decreased (median % drop = 24%; range 13-74%; P < 0.02). One of three individuals at the 10(8) level showed a 39% radiologic decrease in tumor size by cross-section and 75% by volume. HSV infection was documented from liver tumor biopsies. After beginning regional chemotherapy, all patients demonstrated a further decrease in CEA (median 96%; range 50-98%; P < 0.008) and a radiologic partial response. Median survival for this group was 25 months. During follow-up, no signs of virus reactivation were found. Multimutated HSV can be delivered safely into the human bloodstream to produce selective infection of tumor tissues and biologic effects.

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

Therapeutic Efficacy of a Herpes Simplex Virus With Radiation or Temozolomide for Intracranial Glioblastoma After Convection-enhanced Delivery

The herpes simplex virus-1 (HSV-1)-infected cell protein 0 (ICP0) is an E3 ubiquitin ligase implicated in cell cycle arrest and DNA repair inhibition. Convection-enhanced delivery (CED) of either the replication-defective, ICP0-producing HSV-1 mutant, d106, or the recombinant d109, devoid of all viral genome expression, was performed to determine the in vivo efficacy of ICP0 in combination with ionizing radiation (IR) or systemic temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Intracranial U87-MG xenografts were established in athymic nude mice. Animal survival was determined after mice underwent intracranial CED of either the replication-defective d106 or d109 viruses, or Hanks' balanced salt solution (HBSS), before a single session of whole-brain irradiation or TMZ treatment. Median survival for animals that underwent treatment with HBSS alone, d109 alone, d106 alone, HBSS + IR, HBSS + TMZ, d109 + IR, d106 + IR, and d106 + TMZ was 28, 35, 41, 39, 44, 39, 68 (P < 0.01), and 66 days (P < 0.01), respectively. Intracerebral d106 CED resulted in a significant increase in athymic nude mouse survival when combined with IR or TMZ. d106 CED allows for distribution of HSV-1 in human GBM xenografts and persistent viral infection.

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.

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.

Replication-Competent Herpes Simplex Virus 1 Isolates Selected from Cells Transfected with a Bacterial Artificial Chromosome DNA Lacking Only the U L 49 Gene Vary with Respect to the Defect in the U L 41 Gene Encoding Host Shutoff RNase

To generate a null U(L)49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U(L)49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-DeltaU(L)49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U(L)41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U(L)49R DNA (R-U(L)49) accumulated a full-length vhs protein, indicating that in the parental BAC-DeltaU(L)49 DNA, the U(L)41 gene was intact. We conclude that expression of the vhs protein in the absence of U(L)49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U(L)49, to be neutralized.