Herpes Simplex Virus Type 1 Vectors Research Articles

Response to HIV-1 gp160-carrying recombinant virus HSV-1 and HIV-1 VLP combined vaccine in BALB/c mice.

Human immunodeficiency virus (HIV) induced AIDS causes a large number of infections and deaths worldwide every year, still no vaccines are available to prevent infection. Recombinant herpes simplex virus type 1 (HSV-1) vector-based vaccines coding the target proteins of other pathogens have been widely used for disease control. Here, a recombinant virus with HIV-1 gp160 gene integration into the internal reverse (IR) region-deleted HSV-1 vector (HSV-BAC), was obtained by bacterial artificial chromosome (BAC) technology, and its immunogenicity investigated in BALB/c mice. The result showed similar replication ability of the HSV-BAC-based recombinant virus and wild type. Furthermore, humoral and cellular immune response showed superiority of intraperitoneal (IP) administration, compared to intranasally (IN), subcutaneous (SC) and intramuscularly (IM), that evidenced by production of significant antibody and T cell responses. More importantly, in a prime-boost combination study murine model, the recombinant viruses prime followed by HIV-1 VLP boost induced stronger and broader immune responses than single virus or protein vaccination in a similar vaccination regimen. Antibody production was sufficient with huge potential for viral clearance, along with efficient T-cell activation, which were evaluated by the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC). Overall, these findings expose the value of combining different vaccine vectors and modalities to improve immunogenicity and breadth against different HIV-1 antigens.

Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters.

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters’ candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Abstract B23: microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models

Abstract Although oncolytic viruses have shown clinical efficacy for local treatment of cancerous tumors, the ability to induce immune-mediated regression to visceral lesions is not robust. Immune checkpoint modulation has been efficacious in a fraction of cancers associated with an inflamed microenvironment but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies has the exciting potential to be an effective cancer therapy. Oncolytic virus can be genetically modified to decrease pathogenicity to normal cells and to increase their lytic potential and ability to stimulate antitumor immunity, thus improving the risk-benefit ratio for cancer patients. Oncorus oHSV (ONCR) are novel oncolytic herpes simplex virus type-1 vectors developed for the treatment of solid tumors. ONCR utilizes a unique conditional-lethal strategy in which tissue-specific miRs binding sites are inserted into early genes essential for viral replication and in the neurovirulence gene ICP34.5. This strategy curtails viral replication in normal cells, including neurons, and limits the expression of g34.5 to tumor cells, thus promoting potent viral replication in presence of type I interferon. Fifteen immunostimulatory transgenes and combination thereof were systematically evaluated in dual flank syngeneic mouse tumor models to test both for oncolysis and abscopal efficacy on the contralateral noninjected tumor. Among the selected transgenes, IL-12 was found to elicit the most potent efficacy on the ipsilateral and contralateral tumors. We demonstrated that the replication of ONCR and the expression of IL-12 was limited to the injected tumors. By contrast, induction of systemic immunity as assessed by IFNg production in contralateral tumors and in plasma was observed. ONCR expressing IL-12 elicited efficacy in multiple subcutaneous and metastatic models. Additional payloads designed to stimulate antigen presentation, recruitment of immune cells and inhibition of tumor immune suppression were identified as further enhancing IL-12 activity. Data supporting the selection of a suite of payloads cloned into a single ONCR vector will be presented. ONCR represents a new class of oncolytic virus that promote antitumor responses through a multiprong mechanism of action dependent on selective tumor cell killing, the induction of systemic antitumor immunity and reversion of immune suppression. Citation Format: Lorena Lerner, Edward M. Kennedy, Terry Farkaly, Allison Colthart, Caity Montagna, Prajna Behera, Judith Jacques, Peter Grzesik, Jennifer Lee, Agnieszka Denslow, Jacqueline Gursha, Brian Haines, Michael Ball, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Michael Paglia, Laura Viggiano Salta, Lorenz Ponce, Caroline Webb, Mitchell Finer, Christophe Quéva. microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B23.

Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons.

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.

Abstract 4698: ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with poor prognosis and resistance to current therapies. ONCR-1 is a novel oncolytic herpes simplex virus type-1 vector in development for the treatment of GBM. ONCR-1 utilizes a unique conditional-lethal strategy in which miR124 binding sites are inserted into the ICP4 locus to prevent viral replication in neuronal cells while preserving one copy of the γ34.5 gene and enabling potent cytotoxicity in tumor cells. ONCR-1 is armed with a matrix metalloproteinase 9 (MMP9) transgene to facilitate viral spread in the tumor extracellular matrix. Further, ONCR-1 expresses a UL16 binding protein 3 (ULBP3) transgene to activate NK and CD8+ T cells via the NKG2D receptor and promote antitumor immune responses. In human and murine tumor cells in vitro, ONCR-1 evoked potent cytotoxicity. ONCR-1 increased proteolytic MMP in cell supernatants and ULBP3 cell surface expression with no effect on related NKG2D ligands. In mice bearing subcutaneous or orthotopic human U251 GBM tumors, ONCR-1 administered intratumorally (3x105-3x106 PFU QDx1) inhibited tumor growth and prolonged survival. Ex vivo analysis of immune cells in subcutaneous tumors showed significant increases in virally evoked NKG2D+ NK cells with ONCR-1 treatment. In mice bearing dual flank subcutaneous syngeneic A20 tumors, ONCR-1 significantly inhibited tumor growth in injected tumors (1x105-3x105 PFU, Q2Dx3) and evoked potent systemic antitumor immune responses as evidenced by significant inhibition of distal noninjected left flank tumors. IFNγ production from isolated splenocytes cultured in the absence and presence of A20 tumor cells was significantly augmented in ONCR-1-treated animals, providing further evidence of an enhanced antitumor immune response. Moreover, in mice bearing subcutaneous A20 tumors in which ONCR-1 evoked complete tumor regression, complete protection from rechallenge with A20 tumor cells was observed. Depletion of NK cells or CD8+ T cells in mice resulted in inhibition of ONCR-1-mediated antitumor effects in both injected and noninjected murine A20 tumors, supporting an immune mechanism of action. Our preclinical studies demonstrate that ONCR-1 evokes tumor cell killing by direct oncolysis and by enhancing antitumor immune responses. ONCR-1 represents a novel clinical candidate for the treatment of GBM. Citation Format: Alexandra Hicks, Paola Grandi, Michael Paglia, Jingzang Miu, Cecilia Kwong, Jacqueline Gursha, Michael Ball, Weiguo Yao, Daniel Wambua, Terry Farkaly, Kyle Grant, Laura Viggiano Salta, Lorenz Ponce, Joseph Glorioso, Christophe Queva, Mitchell Finer. ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4698.

Synergistic effects of deleting multiple nonessential elements in nonreplicative HSV-1 BAC genomic vectors play a critical role in their viability.

Nonreplicative Herpes simplex virus type-1 (HSV-1) genomic vectors have already entered into clinical trials for neurological gene therapy thanks to their scalable growth in permissive cells. However, the small transgene capacity of this type of HSV-1 vectors currently used in the clinic represents an important limiting factor as a gene delivery system. To develop high-capacity nonreplicative genomic HSV-1 vectors, in this study we have characterized a series of multiply deleted mutants which we have constructed in bacterial artificial chromosomes (BACs), removing up to 24 kb of unstable or dispensable genomic sequences to allow insertion of transgenes up to this size. We show that synergistic effects of deletions of: the HSV-1 replication origins oriS and oriL, the HSV-1 internal repeat region, the remaining ICP4 gene copy and the genes encoding for ICP27, UL56, UL55, can severely reduce the growth of these HSV-1 vectors. Given that several of these elements have been characterized as 'non-essential' for viral growth in cell culture by single-deletion experiments of wild-type HSV-1, our study highlights the need to re-evaluate their functional contribution in the context of multiply deleted nonreplicative HSV-1 genomic vectors. Our BAC mutants described here can serve as useful starting platforms to accelerate HSV-1 vector development.

A haploid HSV-1 genome platform for vector development: testing of the tetracycline-responsive switch shows interference by infected cell protein 0.

Although herpes simplex virus type 1 (HSV-1) has outstanding properties for gene delivery vectors and its genome is available in bacterial artificial chromosomes (BACs) for mutagenesis studies, one impediment is the presence of approximately 15.4kb of DNA sequences that are duplicated in the HSV-1 genome, complicating vector construction and stability. As a useful platform for building HSV-1 vectors, we have constructed a fully haploid HSV-1 genome BAC by deletion of one of these repeats, confirming that viral propagation in culture is not impaired. We used this ΔIR mutant to subsequently investigate whether the insertion of tetracycline-responsive tetO elements into the ICP34.5-ICP0 gene region can be used to control HSV-1 lytic replication. The results of the present study show that ΔIR mutants deleted for ICP34.5 are viable for replication but not when the ICP0 promoter is also disrupted, thus indicating that regulation of infected cell protein 0 (ICP0) levels in the absence of ICP34.5 could be a viable means for controlling growth of HSV-1 vectors. Surprisingly, however, the tetO elements inserted into the ICP0 promoter did not confer ligand responsiveness to growth or ICP0 expression. Further analysis by transfection experiments revealed that ICP0 itself interferes with the tetracycline switch and reduces the the inducibility of this system. Our new haploid HSV-1 BAC is a useful platform for building multiply deleted HSV-1 vectors. Deletion of the gene for ICP34.5 in this backbone renders viral growth dependent on ICP0, although ICP0 expression could not be regulated by tet-responsive transcriptional regulators.

Establishment of a novel therapeutic vector targeting the trigeminal ganglion in rats.

BackgroundIn the pathogenesis of herpes simplex keratitis, herpes simplex virus type 1 (HSV-1) infection begins in corneal epithelium cells and then progresses through the sensory nerve endings and finally travels up forward to the trigeminal ganglion (TG), where it remains as latent virus. The available anti-HSV therapies do not completely suppress the recurrence of active HSV-1 infection. The aim of this study was to establish a novel replication-defective (rd) HSV-1 (rdHSV) vector (rdHSV-interferon gamma [IFNγ]) that could effectively target the TG.MethodsRecombinant HSV-1 virus was inserted into a shuttle plasmid carrying IFNγ to establish the rdHSV-IFNγ vector. Safety was evaluated in vitro by 50% cellular cytotoxicity in transfected SH-SY5Y neuroblastoma cells and in vivo by Kaplan–Meier survival estimate and infection rate. Wistar rats were immunized with rdHSV-IFNγ to evaluate the TG targeting efficiency. Real-time polymerase chain reaction and Western blot assays were used to evaluate IFNγ mRNA and protein expression and rdHSV-IFNγ localization.ResultsThe rdHSV-IFNγ vector was successfully constructed and showed high in vitro safety and overall survival and a corneal infection rate similar to that of control rats immunized with saline (control group; P>0.05). Real-time polymerase chain reaction and immunohistochemistry assays confirmed IFNγ expression and effective TG targeting on days 14 and 21, which increased with postimmunization time. Moreover, IFNγ was expressed sufficiently in the TG tissues.ConclusionThe rdHSV-IFNγ can act as an effective gene transporting vector that carries the therapeutic genes to the TG and triggers its expression.

Dual silencing of Bcl-2 and Survivin by HSV-1 vector shows better antitumor efficacy in higher PKR phosphorylation tumor cells in vitro and in vivo.

RNA interference (RNAi) is a promising tool for cancer therapy, but its delivery strategy is a major challenge for its application. Oncolytic herpes simplex virus type 1 (HSV-1) is not only an effective antitumor drug but also an excellent vector. Herein, RNAi of oncogenes Bcl-2 and Survivin was combined with oncolytic HSV-1 (ICP34.5-/ICP6-/ICP47-/CMV-GM-CSF) and a new vector HSV010-BS was constructed. Transfected cell viability assays and animal experiments revealed that the dual silencing of Bcl-2 and Survivin improved the antitumor effect of oncolytic HSV-1 in vitro and in vivo, while the antitumor effect was correlated with the phosphorylation levels of PKR of the tumor cells. The higher the phosphorylation levels of PKR of the tumor cells, the weaker the replication ability of oncolytic HSV-1, and the more powerful HSV010-BS was than its control vectors in inhibiting the growth of the tumor cells. The results provided direct supportive proofs for a new potential cancer therapy strategy.

Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

Packaging, Amplification, and Appraisal of the Recombinant Tumor-Selective Type I Herpes Simplex Virus Carrying GALV.fus Gene

The aim of the study was to successfully construct three plasmids, which include the GALV.fus gene plasmid regulated by the herpes simplex virus type 1 (HSV-1) late expression gene-UL38 promoter and induced by HSV-1 (HSV-UL38P-GALV.fus), the cytomegalovirus promoter without tumor specificity (CMVP) GALV.fus plasmid (HSV-CMVP-GALV.fus), and the control plasmid in which the GALV.fus gene fragment was replaced by the enhanced green fluorescent protein (EGFP) gene fragment (HSV-CMVP-EGFP). The three constructed plasmids were all packaged and named as Synco-2, Synco-1, and Baco-1. The plasmids were amplified in coliform bacterium and transfected into Vero cells using lipofectamine. These recombinant HSV-1 were amplified in Vero cells and purified by conventional methods of cesium chloride, TCID50 method is used to measure virus titers. The total RNA was then extracted from the HepG2 cells transfected by Synco-1 and Synco-2, and the expression of GALV.fus mRNA was detected by RT-PCR. The three recombinant HSV-1 vectors were propagated in Vero cells and purified by cesium chloride density gradient centrifugation, titrated by TCID50 method, and packaged. The titers of Baco-1, Synco-1, and Synco-2 were 3 × 10(10), 1 × 10(11), and 4 × 10(10) pfu/ml. The GALV.fus gene was identified in the infected HepG2 cells by RT-PCR method.

PD9-07 HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) VECTOR-MEDIATED GENE THERAPY OF ENDOMORPHIN FOR BLADDER OVERACTIVITY AND NOCICEPTION

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2014PD9-07 HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) VECTOR-MEDIATED GENE THERAPY OF ENDOMORPHIN FOR BLADDER OVERACTIVITY AND NOCICEPTION Hiroki Okada, Darren Wolfe, James Wechuck, James R. Goss, Tsuyoshi Majima, Christopher J. Chermansky, and Naoki Yoshimura Hiroki OkadaHiroki Okada More articles by this author , Darren WolfeDarren Wolfe More articles by this author , James WechuckJames Wechuck More articles by this author , James R. GossJames R. Goss More articles by this author , Tsuyoshi MajimaTsuyoshi Majima More articles by this author , Christopher J. ChermanskyChristopher J. Chermansky More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.789AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder pain syndrome/interstitial cystitis (BPS/IC) is a debilitating chronic disorder characterized by bladder pain, urinary frequency and urgency. These symptoms are often difficult to treat and, in such cases, narcotic analgesics that act on μ-opioid receptors (MOR) become the treatment of choice; however, the serious side effects limit their use. Therefore, we examine the effect of local delivery of endomorphin, the natural ligand of MOR, on bladder overactivity and pain behavior using an engineered, non-replicating herpes simplex virus type 1 (HSV-1) vector in rats. METHODS HSV-1 vectors expressing endomorphin (PGN-412) or a placebo vector were injected into the bladder wall of female SD rats 2 weeks prior to the following studies. (1) Cystometry under the awake condition: Saline was continuously infused into the bladder at a rate of 0.04 ml/min to evaluate bladder activity. After a saline infusion period, bladder overactivity was induced by the instillation of 1µM resiniferatoxin (RTX, a TRPV1 receptor agonist) followed by the washout and instillation of 0.5% acetic acid (AA). (2) Behavioral assessment: RTX (3µM, 0.3 ml) was administered into the bladder and retained for 1 min through a temporary indwelled urethral catheter to evaluate nociceptive behaviors such as licking (lower abdominal licking) and freezing (motionless head-turning) for 15 min. RESULTS (1) Inter-contraction intervals (ICI) during cystometry showed a significant reduction after RTX and AA instillation in both PGN-412-treated (n=5) and placebo vector groups (n=4). However, the reduction rate of ICI by RTX and AA in the PGN-412 group was significantly smaller than those in the placebo vector group (RTX: 54±7% vs. 72±10%, AA: 45±12% vs. 76±7%). (2) Intravesical application of RTX for 1 min significantly increased both licking and freezing behaviors in the placebo vector group (n=10); however, these pain-related behaviors were significantly decreased in the PGN-412 group (n=6) compared to the placebo vector group. CONCLUSIONS HSV-1 vector-mediated endomorphin gene therapy targeting the bladder and bladder afferent pathways reduces bladder overactivity and pain-related behavior induced by nociceptive bladder stimuli in rats. Thus, endomorphin gene therapy could be a new treatment modality of urinary frequency and/or bladder pain in patients with BPS/IC with few adverse events. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e214 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Hiroki Okada More articles by this author Darren Wolfe More articles by this author James Wechuck More articles by this author James R. Goss More articles by this author Tsuyoshi Majima More articles by this author Christopher J. Chermansky More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Biosafety of Gene Therapy Vectors Derived From Herpes Simplex Virus Type 1

The majority of humans have been infected with Herpes Simplex Virus Type 1 (HSV-1) and harbor its viral DNA in the latent form within neurons for lifetime. This, combined with the absence of serious adverse effects due to HSV-1 derived vectors in clinical trials so far, highlight the potential to use this virus to develop neuronal gene transfer vectors which are transparent to the host, allowing the effects of the transgene to act without interference from the transfer system eg., for functional genomics in basic neuroscience or gene therapy of neurological disorders. On the other hand, other HSV-1 derived vectors which also have a promising perspective in the clinic, are designed to have enhanced cytotoxicity in certain cell types, as in the case of oncolytic vectors. Understanding virus-host interactions is fundamental not only to the success of these gene therapy vectors but also with respect to identifying and minimizing biohazards associated with their use. In this review we discuss characteristics of HSV-1 and gene therapy vectors derived from this virus which are useful to consider in the context of biosafety risk assessment and risk management.

Immunogenic HSV-mediated Oncolysis Shapes the Antitumor Immune Response and Contributes to Therapeutic Efficacy

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.

Replication-Defective HSV-1 Effectively Targets Trigeminal Ganglion and Inhibits Viral Pathopoiesis by Mediating Interferon Gamma Expression in SH-SY5Y Cells

It has been widely believed that recurrence of herpes simplex keratitis (HSK) is due to the reactivation of herpes simplex virus type 1 (HSV-1) from latent sites in trigeminal ganglion (TG). However, there are also not effective vectors which could target TG for therapy. Replication-defective HSV-1 vector (rdHSV-IFNγ) was established by calcium phosphate co-transfection of complementing cells. We firstly infected rdHSV-IFNγ to SH-SY5Y, and detected IFNγ expression by western blot, evaluated 50 % cellular cytotoxicity (CC(50)) by ELISA. Antiviral activity of rdHSV-IFNγ was examined by immunofluorescence and antiviral concentration of 50 % effectiveness (EC(50)) assay. The rdHSV-IFNγ vector was immunized to Wistar rats to observe targeting function to TG. Kaplan-Meier survival analysis was utilized to assess security of rdHSV-IFNγ. RT-PCR and immunohistochemistry assay were employed to detect rdHSV-IFNγ localization in TG. Western blot was employed to detect IFNγ expression. rdHSV-IFNγ was successfully established, and performed an effective antiviral activity and higher security in SH-SY5Y. There were no significant differences of survival and corneal infection rate of rdHSV-IFNγ immunized rats among groups (P > 0.05). RT-PCR and immunohistochemistry indicated that expression of glycoprotein D (gD) in TG could target TG and decreased following with times post immunization. Furthermore, IFNγ was expressed effectively in TG tissues. Our findings indicated that established rdHSV-IFNγ vector effectively transported therapeutic gene into TG tissues. The administration of replication-defective vector carrying therapeutic genes may become a promising tool in inhibition or reoccurrence of HSK in clinical.

Overview of Gene Delivery into Cells Using HSV‐1‐Based Vectors

This overview describes the considerations involved in the preparation and use of a herpes simplex virus type 1 (HSV-1) amplicon as a vector for gene transfer into neurons. Strategies for gene delivery into neurons, either to study the molecular biology of brain function or for gene therapy, must utilize vectors that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system in vivo. This unit describes the biology of HSV-1 along with a discussion covering development of amplicon and genomic HSV-1 vectors. Advantages and disadvantages of current HSV-1 vectors are presented, and HSV-1 vectors are compared with other vectors for gene transfer into neurons.

A new approach with less damage: intranasal delivery of tetracycline-inducible replication-defective herpes simplex virus type-1 vector to brain

Gene therapy holds great potential for treating neurological disorders. However, delivering gene vectors to the brain has been either invasive or inefficacious in most studies to date. The aim of this study was to develop a safe and efficacious strategy for delivering gene vectors to the brain. A tetracycline-inducible replication-defective herpes simplex virus type-1 vector, QR9TO-LacZ, was administered to rats intranasally. QR9TO-LacZ could infect primary cortical neurons and express the reporter gene without detectable replication. QR9TO-LacZ was observed in the olfactory bulb, hippocampus, striatum, cortex, medulla, cerebellum, ventricles, and nasal septum after intranasal administration. Expression of the reporter gene could be controlled effectively by tetracycline. In vitro, introduction of QR9TO-LacZ did not change the structure of transfected neurons. In vivo, QR9TO-LacZ did not increase apoptosis in neurons and did not alter levels of interleukin 6 and tumor necrosis factor α in the brain after intranasal delivery. Our data suggest that intranasally applied QR9TO-LacZ has a wide distribution and expresses the reporter gene in the brain under the control of tetracycline with less cytotoxicity than intravenous or stereotactic delivery methods.

A novel fusogenic herpes simplex virus for oncolytic virotherapy of squamous cell carcinoma

BackgroundR849 is a neurovirulent γ134.5 gene-deficient form of herpes simplex virus type 1 (HSV-1) and has LacZ genes at the deleted sites of the γ134.5 gene. HF is a spontaneously occurring, fusogenic HSV-1 strain. The purpose of this work was to generate a virus that has the syncytial character of HF, while preserving the γ134.5 gene inactivation profile of R849 virus.ResultsVero cells were infected with R849 and HF simultaneously and two viruses, RH1 and RH2, expressing the LacZ gene and inducing extensive cell fusion were selected. A polymerase chain reaction (PCR)-based analysis suggested that one copy of the γ134.5 gene is lost in RH1, whereas both copies are lost in RH2, and that the γ134.5 gene is replaced by a R849-derived DNA fragment with the LacZ gene. These viruses produced larger plaques and more progeny than the parental viruses. Infection with RH2 decreased the viability of oral squamous cell carcinoma (SCC) cells most strongly. When RH2 was injected into xenografts of oral SCC in nude mice, multinucleated cells were produced and the growth of the tumors was suppressed significantly.ConclusionThese results indicate that novel oncolytic HSV-1 vectors can be produced with the genetic background of the oncolytic HSV-1 HF, and that RH2 is deficient in γ134.5 genes and shows extensive cytopathic effects in oral SCC cells. RH2 may be useful in oncolytic virotherapy for oral SCC.

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.

Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.