RESUMO
BACKGROUND: HSV1790 is an oncolytic virus generated by inserting the enzyme nitroreductase (NTR) into the virus HSV1716. NTR converts the prodrug CB1954 into an active alkylating agent. MATERIALS AND METHODS: In vitro, 3T6 cells (non permissive to HSV) were used in order to distinguish between virus-induced cytopathic effect and cell death due to activated prodrug. In vivo, xenograft models were injected with HSV1790 (10(5)-10(9) PFU) with or without CB1954 (max 80mg/kg) and tumor volume recorded regularly. Biodistribution of HSV1790 was determined immunohistochemically and by PCR. RESULTS: HSV1790 + CB1954 in vitro was more effective at killing tumor cells than the virus or the prodrug alone. In vivo, the combination reduced tumor volume and increased survival compared to treatment with HSV1790 or CB1954 alone. Following systemic administration of HSV1790, viral replication was detected in tumors, but not organs. CONCLUSION: HSV1790 + prodrug enhances tumor cell killing in vitro and reduces tumor volume and increases survival in vivo.
Assuntos
Antineoplásicos/uso terapêutico , Aziridinas/uso terapêutico , Herpesvirus Humano 1/patogenicidade , Neoplasias Experimentais/terapia , Vírus Oncolíticos/metabolismo , Pró-Fármacos/uso terapêutico , Animais , Western Blotting , Terapia Combinada , Feminino , Herpes Simples/genética , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/virologia , Vírus Oncolíticos/genética , Reação em Cadeia da Polimerase , Pró-Fármacos/farmacocinética , Distribuição Tecidual , Replicação ViralRESUMO
GADD34 is expressed in the ischaemic brain and reverses protein synthesis shutdown. Consequently, GADD34 could have neuroprotective potential in stroke. BHK medium, a replication-deficient HSV viral vector (HSV1716) with no insert or containing full-length GADD34, the N terminal or a conserved portion of the gene, was injected into mouse brain before stroke. Infarct size was 1.0+/-0.26, 1.19+/-0.36, 1.5+/-0.36, 1.3+/-0.36, and 1.1+/-0.28 mm3, respectively. The increase in infarct size with full-length GADD34 was statistically significant (P<0.05). Immunohistochemistry confirmed viral protein expression. Tissue culture studies revealed GADD34 gene restored virulence in HSV1716, suggesting that HSV virulence, rather than increased GADD34, exacerbated ischaemic damage.
Assuntos
Antígenos de Diferenciação/genética , Isquemia Encefálica/virologia , Proteínas de Ciclo Celular/genética , Vetores Genéticos/efeitos adversos , Simplexvirus/patogenicidade , Acidente Vascular Cerebral/virologia , Animais , Infarto Encefálico/patologia , Infarto Encefálico/virologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Fosfatase 1 , Simplexvirus/fisiologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologia , VirulênciaRESUMO
BACKGROUND: : Herpes simplex virus (HSV) 1716 is a selectively replicating oncolytic virus. Our objective was to assess the potential efficacy of HSV1716 in patients with oral squamous cell carcinoma (SCC) by intratumoral injection. METHODS: : Twenty patients with oral SCC had a single intratumoral injection of HSV1716 at a dose of 105 pfu (plaque forming unit) or 5 x 105 pfu. Injections were done at 1, 3, or 14 days before surgical resection. The tumors were assessed for evidence of viral replication and necrosis. Immunologic response to virus and toxicity was also assessed. RESULTS: : Intratumoral injections were well tolerated with no adverse effects. Evidence of biological activity was lacking, with no increase in detectable virus in tumor samples. CONCLUSION: : Intratumoral injection of HSV1716 is safe but with little evidence for viral replication or efficacy. Further studies at higher doses are required to determine the potential efficacy of this virus in head and neck cancer.
Assuntos
Carcinoma de Células Escamosas/terapia , Herpesvirus Humano 1 , Neoplasias Bucais/terapia , Terapia Viral Oncolítica , Adulto , Idoso , DNA Viral/isolamento & purificação , Feminino , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Replicação ViralRESUMO
Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.
Assuntos
Herpesvirus Humano 1/fisiologia , Animais , Células CHO , Ciclo Celular/fisiologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células HeLa , Humanos , Especificidade da Espécie , Células VeroRESUMO
BACKGROUND: The neurotropic herpes simplex virus mutant HSV1716 lacks the gene encoding the virulence factor ICP34.5 and cannot replicate in non-dividing cells where proliferating cell nuclear antigen (PCNA) is not actively engaged in cellular DNA synthesis. In the brain, tumoral expression of PCNA therefore confers on it oncolytic specificity and may determine its efficacy. Three phase I trials in glioma patients and one in metastatic melanoma patients have established that HSV1716 is safe and replicates selectively in tumour tissue. Here we examine the in situ PCNA profiles of common human metastatic brain tumours and determine their in vitro permissivity for HSV1716 replication to ascertain their suitability for HSV1716 therapy. METHODS: Histological sections of tumour biopsies obtained from patients undergoing craniotomies were stained for PCNA expression. The replicative ability of HSV wild-type (17(+)) and mutant (1716) viruses was assessed in tissue cultures of the same tumour biopsies and in cancer cell lines by plaque assay. RESULTS: Biopsies of all 10 metastatic tumours (3 melanoma, 4 carcinoma and 3 adenocarcinoma) as well as 4 glioblastoma multiforme were positive for PCNA immunoexpression and supported the replication of HSV1716. The PCNA-positive cells in the metastatic tumours were distributed comparatively in larger and more contiguous areas than in glioblastoma (1.69 +/- 1.61 mm(2) vs. 0.73 +/- 0.77 mm(2)) and numbered 29.0 +/- 12.4 and 12.6 +/- 4.7%, respectively. CONCLUSIONS: The results show that human cerebral metastatic tumours have generally larger and more contiguous proliferative areas, support efficient HSV1716 replication, and are thus potential candidates for such oncolytic viral therapy.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Simplexvirus/fisiologia , Replicação Viral/fisiologia , Adulto , Idoso , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The replicative ability of ICP34.5-null herpes simplex virus (HSV) is cell type and state dependent. In certain cells, ICP34.5 interacts with protein phosphatase 1 to preclude host cell protein synthesis shutoff by dephosphorylation of the eukaryotic initiation factor eIF-2alpha. However, host cell shutoff is not induced by ICP34.5-null HSV in most cells, irrespective of type and state. In general, dividing cells support replication of ICP34.5-null HSV; nondividing cells cannot. Previously the authors showed that ICP34.5 binds to proliferating cell nuclear antigen (PCNA), a protein necessary for cellular DNA replication and repair. Here the authors demonstrate that (1) the interaction between ICP34.5 and PCNA involves two regions of the virus protein; (2) ICP34.5 forms a complex with HSV replication proteins that is DNA binding; (3) at early times in infection, ICP34.5 colocalizes with PCNA and HSV replication proteins in cell nuclei, before accumulating in the cytoplasm; and (4) ICP34.5 is a virion protein. In light of ongoing clinical trials assessing the safety and efficacy of ICP34.5-null HSV, it is vital that the roles of ICP34.5 in HSV replication are understood. The authors propose that in nondividing cells, ICP34.5 is required to switch PCNA from repair to replication mode, a prerequisite for the initiation of HSV replication.