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Abstract Background The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma cell cultures. Results A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis. We analyzed the cytopathic activity of this RL-07 mutant virus. A cell-viability assay showed increased cytopathic activity of the RL-07 mutant virus on U251 and SNB19 glioma cell lines. The plaque sizes of RL-07 on U251 monolayers were seven times larger than those of isogenic control viruses. Similarly, the cytopathic activity of the RL-07 viruses was strongly increased in six primary high-grade glioma cell cultures. In glioma cell lines the RL-07 virus was found to be released earlier into the culture medium. This was not due to enhanced viral protein synthesis, as was evident from equivalent E1A, Fiber and Adenovirus Death Protein amounts, nor to higher virus yields. Conclusion The cytopathic activity of replicating adenovirus in glioblastoma cells is increased by truncating the i-leader open reading frame. Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.

Human adenoviruses (HAdVs) are being explored as vectors for gene transfer and vaccination. Human adenovirus type 26 (HAdV26), which belongs to the largest subgroup of adenoviruses, species D, has a short fiber and a so-far-unknown natural tropism. Due to its low seroprevalence, HAdV26 has been considered a promising vector for the development of vaccines. Despite the fact that the in vivo safety and immunogenicity of HAdV26 have been extensively studied, the basic biology of the virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is poorly understood. In this work, we investigated the roles of the coxsackievirus and adenovirus receptor (CAR), CD46, and αv integrins in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss-offunction studies, we found that αvβ3 integrin is required for efficient infection of epithelial cells by HAdV26, while CAR and CD46 did not increase the transduction efficiency of HAdV26. By studying intracellular trafficking of fluorescently labeled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and that increased αvβ3 integrin enhances internalization of HAdV26. Thus, we conclude that HAdV26 uses αvβ3 integrin as a receptor for infecting epithelial cells. These results give us new insight into the HAdV26 infection pathway and will be helpful in further defining HAdV-based vector manufacturing and vaccination strategies. IMPORTANCE Adenovirus-based vectors are used today for gene transfer and vaccination. HAdV26 has emerged as a promising candidate vector for development of vaccines due to its relatively low seroprevalence and its ability to induce potent immune responses against inserted transgenes. However, data regarding the basic biology of the virus, like receptor usage or intracellular trafficking, are limited. In this work, we found that efficient infection of human epithelial cell lines by HAdV26 requires the expression of the αvβ3 integrin. By studying intracellular trafficking of fluorescently labeled HAdV26 in a cell clone with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and confirmed that αvβ3 integrin expression facilitates efficient HAdV26 internalization. These results will allow further improvement of HAdV26-based vectors for gene transfer and vaccination. [ABSTRACT FROM AUTHOR]

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV. [ABSTRACT FROM AUTHOR]

Background: The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma cell cultures. Results: A mutation truncating the i-leader open reading frame was created in a molecular clone of replicationcompetent wild-type HAdV-5 by site-directed mutagenesis. We analyzed the cytopathic activity of this RL-07 mutant virus. A cell-viability assay showed increased cytopathic activity of the RL-07 mutant virus on U251 and SNB19 glioma cell lines. The plaque sizes of RL-07 on U251 monolayers were seven times larger than those of isogenic control viruses. Similarly, the cytopathic activity of the RL-07 viruses was strongly increased in six primary high-grade glioma cell cultures. In glioma cell lines the RL-07 virus was found to be released earlier into the culture medium. This was not due to enhanced viral protein synthesis, as was evident from equivalent E1A, Fiber and Adenovirus Death Protein amounts, nor to higher virus yields. Conclusion: The cytopathic activity of replicating adenovirus in glioblastoma cells is increased by truncating the ileader open reading frame. Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma. [ABSTRACT FROM AUTHOR]

Background Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context. Methods Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers. Results In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent. Conclusions LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

An abstract of the article "Toward 'Pseudotyping' Adenovirus Vectors: Efficient Generation of pIX-Expressing Helper Cell Lines," by Rob C. Hoeben, Taco G. Uil, Jeroen De Vrij, Leif Lindholm and Jort Vellinga is presented.

Abstract: In human adenoviruses (HAdV), 240 copies of the 14.3-kDa minor capsid protein IX stabilize the capsid. Three N-terminal domains of protein IX form triskelions between hexon capsomers. The C-terminal domains of four protein IX monomers associate near the facet periphery. The precise biological role of protein IX remains enigmatic. Here we show that deletion of the protein IX gene from a HAdV-5 vector enhanced the reporter gene delivery 5 to 25-fold, specifically to Coxsackie and Adenovirus Receptor (CAR)-negative cell lines. Deletion of the protein IX gene also resulted in enhanced activation of peripheral blood mononuclear cells. The mechanism for the enhanced transduction is obscure. No differences in fiber loading, integrin-dependency of transduction, or factor-X binding could be established between protein IX-containing and protein IX-deficient particles. Our data suggest that protein IX can affect the cell tropism of HAdV-5, and may function to dampen the innate immune responses against HAdV particles. [ABSTRACT FROM AUTHOR]

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. [Copyright &y& Elsevier]

Oncolytic viruses that are replication competent in tumor but not in normal cells represent a novel approach for treating neoplastic diseases. However, the oncolytic potency of replicating agents is determined directly by their capability of infecting target cells. Most adenoviruses used for gene therapy or virotherapy have been based on serotype 5 (Ad5). Unfortunately, expression of the primary receptor for Ad5 (the coxsackie–adenovirus receptor, or CAR) is highly variable on ovarian and other cancer cells. By performing genetic fiber pseudotyping, we created Ad5/3-Δ24, a conditionally replicating adenovirus that does not bind CAR but facilitates entry into and killing of ovarian cancer cells. We show replication of Ad5/3-Δ24 and subsequent oncolysis of ovarian adenocarcinoma lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell spheroids purified from patient samples. Moreover, in a therapeutic orthotopic model of peritoneal carcinomatosis, dramatically enhanced survival was noted. Finally, Ad5/3-Δ24 achieved a significant antitumor effect as assessed by noninvasive, in vivo bioluminescence imaging. Therefore, the preclinical therapeutic efficacy of Ad5/3-Δ24 is improved over the respective CAR- and integrin-binding controls. Taken together with promising biodistribution and toxicity data, this approach could translate into successful clinical interventions for ovarian cancer patients. [Copyright &y& Elsevier]

Human adenoviruses (HAdVs) are being explored as vectors for gene transfer and vaccination. Human adenovirus type 26 (HAdV26), which belongs to the largest subgroup of adenoviruses, species D, has a short fiber and a so-far-unknown natural tropism. Due to its low seroprevalence, HAdV26 has been considered a promising vector for the development of vaccines. Despite the fact that the in vivo safety and immunogenicity of HAdV26 have been extensively studied, the basic biology of the virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is poorly understood. In this work, we investigated the roles of the coxsackievirus and adenovirus receptor (CAR), CD46, and αv integrins in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss-of-function studies, we found that αvβ3 integrin is required for efficient infection of epithelial cells by HAdV26, while CAR and CD46 did not increase the transduction efficiency of HAdV26. By studying intracellular trafficking of fluorescently labeled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and that increased αvβ3 integrin enhances internalization of HAdV26. Thus, we conclude that HAdV26 uses αvβ3 integrin as a receptor for infecting epithelial cells. These results give us new insight into the HAdV26 infection pathway and will be helpful in further defining HAdV-based vector manufacturing and vaccination strategies. IMPORTANCE Adenovirus-based vectors are used today for gene transfer and vaccination. HAdV26 has emerged as a promising candidate vector for development of vaccines due to its relatively low seroprevalence and its ability to induce potent immune responses against inserted transgenes. However, data regarding the basic biology of the virus, like receptor usage or intracellular trafficking, are limited. In this work, we found that efficient infection of human epithelial cell lines by HAdV26 requires the expression of the αvβ3 integrin. By studying intracellular trafficking of fluorescently labeled HAdV26 in a cell clone with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and confirmed that αvβ3 integrin expression facilitates efficient HAdV26 internalization. These results will allow further improvement of HAdV26-based vectors for gene transfer and vaccination., (Copyright © 2018 American Society for Microbiology.)

Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease.

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.