Genome Analyses of Ten New Ape Adenoviruses with Similarity to Human Mastadenovirus C.

The adenoviruses (AdVs) isolated from humans are taxonomically grouped in seven different species in the Mastadenovirus genus (HAdV-A through G). AdVs isolated from apes are often included in one of the human AdV species. Here we describe the sequence analyses of ten new AdVs that are related to the HAdV-C species and that were isolated from healthy western lowland gorillas, bonobos, chimpanzees, and orangutans kept in Dutch zoos. We analyzed these viruses and compared their genome sequences to those of human- and ape-derived AdV sequences in the NCBI GenBank database. Our data demonstrated that the ape-derived viruses clustering to HAdV-C are markedly distinct from the human HAdV-C species in the size and nucleotide composition (%GC) of their genome, differ in the amino-acid sequence of AdV proteins, and have longer RGD-loops in their penton-base proteins. The viruses form three well-separated clades (the human, the gorilla, and the combined group of the bonobo and chimpanzee viruses), and we propose that these should each be given species-level ranks. The Ad-lumc005 AdV isolated from orangutans was found to be very similar to the gorilla AdVs, and bootstrap inference provided evidence of recombination between the orangutan AdV and the gorilla AdVs. This suggests that this virus may not be a genuine orangutan AdV but may have been transferred from a gorilla to an orangutan host.

The stability of envelope-pseudotyped lentiviral vectors.

Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the 'Reduction Ratio.' This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector's envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2-110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.

NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice-A Novel in Vivo Imaging Platform.

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

Characterization of a replicating expanded tropism oncolytic reovirus carrying the adenovirus E4orf4 gene.

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.

Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells.

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus' tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two- and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus' capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.

Oncolytic Reovirus Infection Is Facilitated by the Autophagic Machinery.

Mammalian reovirus is a double-stranded RNA virus that selectively infects and lyses transformed cells, making it an attractive oncolytic agent. Despite clinical evidence for anti-tumor activity, its efficacy as a stand-alone therapy remains to be improved. The success of future trials can be greatly influenced by the identification and the regulation of the cellular pathways that are important for reovirus replication and oncolysis. Here, we demonstrate that reovirus induces autophagy in several cell lines, evident from the formation of Atg5-Atg12 complexes, microtubule-associated protein 1 light chain 3 (LC3) lipidation, p62 degradation, the appearance of acidic vesicular organelles, and LC3 puncta. Furthermore, in electron microscopic images of reovirus-infected cells, autophagosomes were observed without evident association with viral factories. Using UV-inactivated reovirus, we demonstrate that a productive reovirus infection facilitates the induction of autophagy. Importantly, knock-out cell lines for specific autophagy-related genes revealed that the expression of Atg3 and Atg5 but not Atg13 facilitates reovirus replication. These findings highlight a central and Atg13-independent role for the autophagy machinery in facilitating reovirus infection and contribute to a better understanding of reovirus-host interactions.

Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

Enhanced transduction of CAR-negative cells by protein IX-gene deleted adenovirus 5 vectors.

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.

Adenovirus-derived vectors for prostate cancer gene therapy.

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.

Clinical adenoviral gene therapy for prostate cancer.

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.

Modification of mammalian reoviruses for use as oncolytic agents.

The Reoviridae are a family of viruses with a non-enveloped icosahedral capsid and a segmented double-stranded RNA genome. Prototypes of the mammalian Orthoreoviruses have been isolated from human respiratory and enteric tracts and are not associated with human disease. One of these, human reovirus type 3 Dearing (T3D), usually serves as a model for the family. In the last decade the mammalian Orthoreoviruses, especially T3D, have been evaluated as oncolytic agents in experimental cancer therapy. This is based on the observation that reoviruses induce cell death and apoptosis in tumor cells, but not in healthy non-transformed cells. Several clinical trials have been initiated in Canada, the USA, and the UK, to study the feasibility and safety of this approach. Due to the segmented structure of their double-stranded RNA genomes genetic modification of Reoviridae has been notoriously difficult. Several techniques have been described recently that facilitate the genetic modification of reovirus genomes. The basis for reverse genetics of reovirus is the discovery in 1990 that reovirus RNA is infectious. Subsequently, it took ten years before a foreign gene was introduced into the reovirus genome. Here we review the methods for reovirus modification and their use for generating new reovirus-derived oncolytic agents.