Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEÄ), thymidine kinase (BoHV-5TKÄ) and both proteins (BoHV-5gEÄTKÄ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEÄ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKÄ (N = 8) or BoHV-5gEÄTKÄ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKÄ and BoHV-5gEÄTKÄ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy

PURPOSE: We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. MATERIALS AND METHODS: Replication-competent recombinant adenoviral vector (Ad-deltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-deltaE1A) was generated as a control. Both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. RESULTS: On gamma counter-analyses, counts/minute (cpm)/microgram of protein showed MOIs dependency with deltaE1B19/55-TK infection. On MTT assay, Ad-deltaE1B19/55-TK led to more efficient cell killing than Ad-deltaE1A-TK. On plate imaging by gamma camera, both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of deltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-deltaE1A-TK infection. CONCLUSION: Replicating Ad-deltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-deltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-deltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.

Increased Anti-tumor Effect by a Combination of HSV Thymidine Kinase Suicide Gene Therapy and Interferon-gamma/GM-CSF Cytokine Gene Therapy in CT26 Tumor Model

The potential therapeutic benefit of introducing IFN-gamma and GM-CSF genes in combination with the HSVtk suicide gene into subcutaneously implanted CT26 tumor cells was compared with that from each treatment alone. Cells, unmodified or retrovirally transduced with HSVtk or IFN-gamma/GM-CSF genes, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSVtk gene, with intraperitoneal ganciclovir treatment, reduced tumor volume by 81% at locally inoculated tumor sites (p<0.01) and by 25% at distantly inoculated tumor sites (p=0.052). IFN-gamma/GM-CSF genes showed a 56% tumor volume reduction at local tumor sites (p<0.01) and 15% volume reduction at remote tumor sites, although this was not statistically significant. The combination of HSVtk (with GCV) and IFN-gamma/GM-CSF genes showed an 81% volume reduction at local tumor sites (p<0.01) and a 43% volume reduction at remote tumor sites (p<0.01). Thus, the combination of HSVtk and IFN-gamma/GM-CSF gene therapy produced greater therapeutic efficacy than either treatment alone.

Bystander-Mediated Regression of Murine Neuroblastoma via Retroviral Transfer of the HSV-TK Gene

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirusmediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.

Terapia genica antitumoral mediante el uso de genes suicidas / Cancer gene therapy using suicide genes

Células tumorales transducidas con vectores retrovirales portanto el gen de la timidina kinasa del virus herpes simplex-1 (HSV-tk), son capaces de transformar la droga antiherpética ganciclovir (GCV) en un metabolito tóxico para células en división. Esta terapia suicida aumenta su eficiencia debido a un efecto "bystander" que induce la muerte de células no transducidas, vecinas a células modificadas. El mecanismo del mencionado efecto no se conoce totalmente, pero existe evidencia que asigna un rol preponderante al sistema inmune, para lograr una completa erradicación tumoral. En este trabajo estudiamos la efectividad del sistema en tres líneas celulares: un melanoma humano y uno murino, y un glioma de rata. Los tumores fueron estabelecidos por inyección de células tumorales s.c. en ratones nude y C57BI/6, e intracerebralmente por esterotaxis en ratas Sprague Dawley, respectivamente. Animales tratados fueron co-inyectados con células productoras de retrovirus expresando HSV-tk y posterior administración i.p. de GCV. En experimentos in vivo a corto plazo, se observó inhibición total o parcial del crecimiento tumoral en todos los modelos. En experimentos de supervivencia a largo plazo con células C6, el 50 por ciento de los animales sobrevivió más de 75 dias (p < 0 0001) y fue capaz de rechazar un desafio con células parentales C6 inyectadas en el hemisferio contralateral. El análisis histológico e inmunohistoquímico mostró la presencia de un infiltrado inflamatorio compuesto por linfocitos T, macrófagos y polimorfonucleares. Estos resultados demuestran que el uso de genes suicidas puede ser una herramienta de enorme importancia en el tratamiento de tumores de cerebro y de metástasis cerebrales.