Preclinical Testing of an Oncolytic Parvovirus in Ewing Sarcoma: Protoparvovirus H-1 Induces Apoptosis and Lytic Infection In Vitro but Fails to Improve Survival In Vivo.

About 70% of all Ewing sarcoma (EWS) patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV) to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU)/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Pediatric and Adult High-Grade Glioma Stem Cell Culture Models Are Permissive to Lytic Infection with Parvovirus H-1.

Combining virus-induced cytotoxic and immunotherapeutic effects, oncolytic virotherapy represents a promising therapeutic approach for high-grade glioma (HGG). A clinical trial has recently provided evidence for the clinical safety of the oncolytic parvovirus H-1 (H-1PV) in adult glioblastoma relapse patients. The present study assesses the efficacy of H-1PV in eliminating HGG initiating cells. H-1PV was able to enter and to transduce all HGG neurosphere culture models (n = 6), including cultures derived from adult glioblastoma, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Cytotoxic effects induced by the virus have been observed in all HGG neurospheres at half maximal inhibitory concentration (IC50) doses of input virus between 1 and 10 plaque forming units per cell. H-1PV infection at this dose range was able to prevent tumorigenicity of NCH421k glioblastoma multiforme (GBM) "stem-like" cells in NOD/SCID mice. Interestingly NCH421R, an isogenic subclone with equal capacity of xenograft formation, but resistant to H-1PV infection could be isolated from the parental NCH421k culture. To reveal changes in gene expression associated with H-1PV resistance we performed a comparative gene expression analysis in these subclones. Several dysregulated genes encoding receptor proteins, endocytosis factors or regulators innate antiviral responses were identified and represent intriguing candidates for to further study molecular mechanisms of H-1PV resistance.

Oncolytic effects of parvovirus H-1 in medulloblastoma are associated with repression of master regulators of early neurogenesis.

Based on extensive pre-clinical studies, the oncolytic parvovirus H-1 (H-1PV) is currently applied to patients with recurrent glioblastoma in a phase I/IIa clinical trial (ParvOryx01, NCT01301430). Cure rates of about 40% in pediatric high-risk medulloblastoma (MB) patients also indicate the need of new therapeutic approaches. In order to prepare a future application of oncolytic parvovirotherapy to MB, the present study preclinically evaluates the cytotoxic efficacy of H-1PV on MB cells in vitro and characterizes cellular target genes involved in this effect. Six MB cell lines were analyzed by whole genome oligonucleotide microarrays after treatment and the results were matched to known molecular and cytogenetic risk factors. In contrast to non-transformed infant astrocytes and neurons, in five out of six MB cell lines lytic H-1PV infection and efficient viral replication could be demonstrated. The cytotoxic effects induced by H-1PV were observed at LD50s below 0.05 p. f. u. per cell indicating high susceptibility. Gene expression patterns in the responsive MB cell lines allowed the identification of candidate target genes mediating the cytotoxic effects of H-1PV. H-1PV induced down-regulation of key regulators of early neurogenesis shown to confer poor prognosis in MB such as ZIC1, FOXG1B, MYC, and NFIA. In MB cell lines with genomic amplification of MYC, expression of MYC was the single gene most significantly repressed after H-1PV infection. H-1PV virotherapy may be a promising treatment approach for MB since it targets genes of functional relevance and induces cell death at very low titers of input virus.

Regression of glioma in rat models by intranasal application of parvovirus h-1.

PURPOSE: In previous studies, we have shown that the apathogenic rat parvovirus H-1 (H-1PV) is capable to induce regression of advanced symptomatic rat and human gliomas in a rat model, when the virus was injected in the tumor (intracranially) or intravenously. Infection with H-1PV did not provoke any pathology in nontumor tissue. This study addresses the question whether also intranasal application of this oncolytic virus is suitable and sufficient for treating gliomas in this animal model. EXPERIMENTAL DESIGN: Rat (RG-2) or human (U87) glioma cells were grafted stereotactically in the brain of rats (Wistar or RNU, respectively), and after development of tumors visible by MRI, H-1PV was instilled intranasally. Tumor regression was monitored by MRI, and survival was analyzed by Kaplan-Meier analysis. Brains from sacrificed animals were analyzed for histologic alterations, presence of viral DNA and proteins and infectious virions. In addition, distribution of virus to other organs was determined. RESULTS: A single intranasal instillation of H-1PV was sufficient to induce efficient regression of rat glioma, leading to significant prolongation of survival without any toxicity for other tissues. It is shown that the virus reaches brain and other tissues, and that the viral replication-associated (and oncolysis-associated) regulatory proteins are exclusively expressed in the tumor tissue. In rats with xenografts of human glioma, oncolytic activity of H-1PV was less pronounced, however, leading to significant prolongation of survival. CONCLUSION: In view of an ongoing clinical trial on the use of H-1PV for oncolytic virotherapy of glioma, the option of applying the virus intranasally may be a valuable alternative to invasive routes of infection.

Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models.

Oncolytic virotherapy is a potential treatment modality under investigation for various malignancies including malignant brain tumors. Unlike some other natural or modified viruses that show oncolytic activity against cerebral neoplasms, the rodent parvovirus H-1 (H-1PV) is completely apathogenic in humans. H-1PV efficiently kills a number of tumor cells without harm to corresponding normal ones. In this study, the concept of H-1PV-based virotherapy of glioma was tested for rat (RG-2 cell-derived) and for human (U87 cell-derived) gliomas in immunocompetent and immunodeficient rat models, respectively. Large orthotopic rat and human glioma cell-derived tumors were treated with either single stereotactic intratumoral or multiple intravenous (iv) H-1PV injections. Oncolysis was monitored by magnetic resonance imaging and proven by histology. Virus distribution and replication were determined in brain and organs. In immunocompetent rats bearing RG-2-derived tumors, a single stereotactic intratumoral injection of H-1PV and multiple systemic (iv) applications of the virus were sufficient for remission of advanced and even symptomatic intracranial gliomas without damaging normal brain tissue or other organs. H-1PV therapy resulted in significantly improved survival (Kaplan-Meier analysis) in both the rat and human glioma models. Virus replication in tumors indicated a contribution of secondary infection by progeny virus to the efficiency of oncolysis. Virus replication was restricted to tumors, although H-1PV DNA could be detected transiently in adjacent or remote normal brain tissue and in noncerebral tissues. The results presented here and the innocuousness of H-1PV for humans argue for the use of H-1PV as a powerful means to perform oncolytic therapy of malignant gliomas.

Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells.

Despite multimodal therapeutic concepts, advanced localized and high-risk neuroblastoma remains a therapeutic challenge with a long-term survival rate below 50%. Consequently, new modalities for the treatment of neuroblastoma, e.g., oncolytic virotherapy are urgently required. H-1PV is a rodent parvovirus devoid of relevant pathogenic effects in infected adult animals. In contrast, the virus has oncolytic properties and is particularly cytotoxic for transformed or tumor-derived cells of various species including cells of human origin. Here, a preclinical in vitro assessment of the application of oncolytic H-1PV for the treatment of neuroblastoma cells was performed. Infection efficiency, viral replication and lytic activity of H-1PV were analyzed in 11 neuroblastoma cell lines with different MYCN status. Oncoselectivity of the virus was confirmed by the infection of short term cultures of nonmalignant infant cells of different origin. In these nontransformed cells, no effect of H-1PV on viability or morphology of the cells was observed. In contrast, a lytic infection was induced in all neuroblastoma cell lines examined at MOIs between 0.001 and 10 pfu/cell. H-1PV actively replicated with virus titres increasing up to 5,000-fold within 48-96 hr after infection. The lytic effect of H-1PV was observed independent of MYCN oncogene amplification or differentiation status. Moreover, a significant G2-arrest and induction of apoptosis could be demonstrated. Infection efficiency, rapid virus replication and exhaustive lytic effects on neuroblastoma cells together with the low toxicity of H-1PV for nontransformed cells, render this parvovirus a promising candidate for oncolytic virotherapy of neuroblastoma.

Cytomegalovirus (CMV) infection--related to male and/or female infertility factors?

OBJECTIVE: To screen for cytomegalovirus (CMV) infection in male and female partners of subfertile couples and to analyze a potential relationship with clinically relevant parameters of male and female factor fertility. DESIGN: Prospective study. SETTING: Outpatient infertility clinic of a university-based hospital. PATIENT(S): Randomly selected male and female partners of asymptomatic subfertile couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Screening for CMV in the semen of the male partner (using nested polymerase chain reaction) and, at the same time, in the endocervical material of the female partner; same-day CMV serology (anti-CMV IgG and IgM class antibodies [Ab]); medical history and clinical examination; evaluation of semen quality, including sperm functional capacity and detection of antisperm Ab (ASA) and seminal white blood cells (WBC) in aliquots of the same ejaculates; bacterial screening of both partners; and detailed examination of the cervical factor and other variables of female subfertility. RESULT(S): The presence of CMV in semen was not significantly related to semen quality including sperm functional capacity, local antisperm Ab, or seminal WBC. CMV in endocervical material was not associated with a reduced quality of the cervical mucus or with other female infertility factors. CMV in semen was not associated with the presence of CMV in the endocervical material of the female partners. CMV infection was not significantly associated with other microorganisms of the lower genital tract. CMV serology (IgG and/or IgM Ab) did not sufficiently reflect CMV presence in semen or the cervix. CONCLUSION(S): CMV presence in the genital tract of subfertile patients is considerable, but findings do not suggest that sexual transmission is a frequent route of infection or that CMV infection is a significant cause of infertility.

Evidence of chromosomal integration of AAV DNA in human testis tissue.

Persistent infection with adeno-associated virus (AAV) has been demonstrated in human tissues, most frequently in the female and male genital tract. The clinical significance of latent AAV infection remains, however, uncertain to date. The mode of latency of AAV is not known, i.e., it is unclear whether the viral genome is integrated in the cellular genome, and if integration occurs site-specifically in chromosome 19 as has been observed in cell culture. Therefore we investigated if viral DNA in AAV DNA-positive human testis samples from two patients, is integrated in the cellular genome. Using two different molecular approaches, uni-directional PCR and Walking Primer PCR, we could demonstrate that AAV DNA is present in an integrated form in testis tissue. Virus-cell DNA junction fragments were cloned and sequenced. A detailed analysis revealed integration within sequences of the so-called AAVS1 region on chromosome 19. These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases.

Sensitization of sarcoma cells to doxorubicin treatment by concomitant wild-type adeno-associated virus type 2 (AAV-2) infection.

Doxorubicin-based chemotherapy is used in the treatment of sarcomas. Toxic side effects and poor response rates underline the demand for an improvement in current chemotherapeutic protocols. Recently, it has been reported that parvoviruses confer various antineoplastic properties to infected cells, and that adeno-associated virus type 2 (AAV-2) infection sensitizes malignant epithelial cells to radiation- or chemotherapy-based genotoxic treatment. Thus, we analyzed whether AAV-2 infection leads to an improved efficacy of doxorubicin chemotherapy in malignant mesenchymal cells, using 13 human sarcoma cell lines. Therapeutic effects were analyzed by measuring cell viability and proliferation (WST-1, colony forming, and propidium iodide assays). Additionally, permissivity for AAV-2 infection was determined by Southern dot blot analysis. AAV-2 infection strongly increased the efficacy of doxorubicin treatment in rhabdomyo-, fibro-, osteo- and chondrosarcoma cells in a dose-dependent manner. This effect was not observed in liposarcoma and synovial sarcoma cells, although a susceptability to AAV-2 infection was documented. Our results indicate that the sensitization effects towards genotoxic treatment exerted by non-pathogenic AAV-2 infection are not restricted to epithelial malignancies but may also be exploited for the improvement of chemotherapy in patients suffering from rhabdomyo-, fibro-, osteo-, or chondrosarcomas.

Adenoassociated virus type 2-induced inhibition of the human papillomavirus type 18 promoter in transgenic mice.

The epithelium of the cervix uteri has been reported to be frequently coinfected with both human papillomaviruses (HPV) and helper virus-dependent adenoassociated viruses (AAV). Seroepidemiological data suggest that AAV infection could inhibit cervical cancer that is caused by specific ("high-risk") types of papillomaviruses. In vitro, infection with AAV type 2 (AAV-2) or transfection of AAV-2 early (rep) genes has been shown to inhibit transformation by papillomaviruses. To analyze the effects of AAV on HPV in vivo, we studied the influence of AAV-2 infection on the promoter activity of high-risk HPV type 18 (HPV-18) in mice, transgenic for sequences of the upstream regulatory region (URR) of HPV-18 controlling transcription of the reporter gene, lacZ. Transgenic animals (or tongue cells thereof, explanted and grown in culture) were treated with dexamethasone to induce the HPV-18 promoter. Simultaneously they were (i) infected with AAV, (ii) inoculated with AAV virus-like particles (VLPs; empty capsids), or (iii) mock infected. Inoculation with AAV-2 or VLPs inhibited activation of the HPV-18 promoter. In vitro, in baby hamster kidney cells transfected with the HPV-18-lacZ construct, tissue extracts from AAV-infected animals suppressed the HPV-18 URR to a similar extent as AAV infection did. Down-regulation of the HPV-18 promoter was less efficient with extracts from animals inoculated with VLPs and was not observed with extracts from uninfected or dexamethasone-treated animals. This indicates that AAV induces cellular factor(s) in vivo capable of mediating down-regulation of the HPV-18 promoter also in cells in vitro. In contrast, promoters of the low-risk HPV types (HPV-6, HPV-11) were not influenced by AAV infection as opposed to promoters of the high-risk types (HPV-18 and HPV-16).

Enhancement of cisplatin-induced apoptosis by infection with adeno-associated virus type 2.

The non-pathogenic human adeno-associated virus, AAV, has been shown to sensitize human cancer cells and experimental tumors towards the action of chemotherapeutic agents such as cisplatin. Since chemotherapeutic drugs mainly involve the induction of apoptosis, we investigated whether 1 possible mechanism of AAV-mediated sensitization of human tumor cells may result from an enhancement of cisplatin-induced apoptosis. In HeLa and A549 cells, infection with AAV type 2 (AAV-2) increased cisplatin-induced DNA fragmentation but had no cytotoxic effect by itself. This enhanced apoptosis appeared to be mediated at least in part by a component of the viral capsid since empty or UV-inactivated AAV-2 particles were also able to boost cisplatin-induced DNA fragmentation. Interestingly, these effects were not observed after infection with AAV type 5 (AAV-5) or the autonomous parvovirus, H-1. AAV-2-mediated enhancement of apoptosis was not associated with a modification of the expression of CD95 ligand, CD95 receptor or other death receptors, as shown by RT-PCR and RNase protection assay. In contrast, using the mitochondrial fluorescent dye, JC-1 in flow cytometry, AAV-2 infection was found to further reduce the mitochondrial transmembrane potential after treatment with cisplatin in a caspase-independent manner, suggesting that increase of apoptosis by AAV-2 occurred at the mitochondrial level. In contrast, in cells of the small cell lung cancer line, P693, an enhancement of cisplatin-induced DNA fragmentation was not observed after infection with AAV-2. In these cells, sensitization to cisplatin-toxicity was associated with cell cycle arrest in G2/M. The data indicate that in the absence of viral gene expression, AAV-2-mediated sensitization to cisplatin involves multiple cellular pathways promoting cell death signals in a cell type-dependent manner. The results further support that AAV-2 particles may be appropriate adjuvants for improving cancer chemotherapy and may also have consequences regarding AAV-2-based vectors for gene therapy.