Replication-competent human adenovirus 11p vectors can propagate in Vero cells

International Nuclear Information System (INIS)

Gokumakulapalle, Madhuri; Mei, Ya-Fang

2016-01-01

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

Replication-competent human adenovirus 11p vectors can propagate in Vero cells

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Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

2016-08-15

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

Core labeling of adenovirus with EGFP

International Nuclear Information System (INIS)

Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T.

2006-01-01

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology

Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications

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Hanni Uusi-Kerttula

2015-11-01

Full Text Available Adenoviruses (Ad are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies.

Production and purification of non replicative canine adenovirus type 2 derived vectors.

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Szelechowski, Marion; Bergeron, Corinne; Gonzalez-Dunia, Daniel; Klonjkowski, Bernard

2013-12-03

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.

Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

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Sandy Ibanes

Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

Canine Adenovirus Type 2 Vector Generation via I-Sce1-Mediated Intracellular Genome Release

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Ibanes, Sandy; Kremer, Eric J.

2013-01-01

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation. PMID:23936483

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production

OpenAIRE

Nuno Carinhas; Daniel A. M. Pais; Alexey Koshkin; Paulo Fernandes; Ana S. Coroadinha; Manuel J. T. Carrondo; Paula M. Alves; Ana P. Teixeira

2016-01-01

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-13C]glucose and [U-13C]glutamine, we apply for the first time 13C-Metabolic flux analysis (13C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells meta...

Immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity

NARCIS (Netherlands)

Thorner, Anna R.; Lemckert, Angelique A. C.; Goudsmit, Jaap; Lynch, Diana M.; Ewald, Bonnie A.; Denholtz, Matthew; Havenga, Menzo J. E.; Barouch, Dan H.

2006-01-01

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have

A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

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Gallaher, Sean D.; Berk, Arnold J.

2013-01-01

Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity. PMID:23624118

Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures.

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Di Yu

Full Text Available Recombinant adenovirus serotype 5 (Ad5 vectors represent one of the most efficient gene delivery vectors in life sciences. However, Ad5 is dependent on expression of the coxsackievirus-adenovirus-receptor (CAR on the surface of target cell for efficient transduction, which limits it's utility for certain cell types. Herein we present a new vector, Ad5PTDf35, which is an Ad5 vector having serotype 35 fiber-specificity and Tat-PTD hexon-modification. This vector shows dramatically increased transduction capacity of primary human cell cultures including T cells, monocytes, macrophages, dendritic cells, pancreatic islets and exocrine cells, mesenchymal stem cells and tumor initiating cells. Biodistribution in mice following systemic administration (tail-vein injection show significantly reduced uptake in the liver and spleen of Ad5PTDf35 compared to unmodified Ad5. Therefore, replication-competent viruses with these modifications may be further developed as oncolytic agents for cancer therapy. User-friendly backbone plasmids containing these modifications were developed for compatibility to the AdEasy-system to facilitate the development of surface-modified adenoviruses for gene delivery to difficult-to-transduce cells in basic, pre-clinical and clinical research.

Improvement of oncolytic adenovirus vectors through genetic capsid modifications

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Vrij, Jeroen de

2012-01-01

Recombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are among the most popular of viruses to be developed. Although clinical studies have demonstrated safety of

Single-cycle adenovirus vectors in the current vaccine landscape.

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Barry, Michael

2018-02-01

Traditional inactivated and protein vaccines generate strong antibodies, but struggle to generate T cell responses. Attenuated pathogen vaccines generate both, but risk causing the disease they aim to prevent. Newer gene-based vaccines drive both responses and avoid the risk of infection. While these replication-defective (RD) vaccines work well in small animals, they can be weak in humans because they do not replicate antigen genes like more potent replication-competent (RC) vaccines. RC vaccines generate substantially stronger immune responses, but also risk causing their own infections. To circumvent these problems, we developed single-cycle adenovirus (SC-Ad) vectors that amplify vaccine genes, but that avoid the risk of infection. This review will discuss these vectors and their prospects for use as vaccines. Areas covered: This review provides a background of different types of vaccines. The benefits of gene-based vaccines and their ability to replicate antigen genes are described. Adenovirus vectors are discussed and compared to other vaccine types. Replication-defective, single-cycle, and replication-competent Ad vaccines are compared. Expert commentary: The potential utility of these vaccines are discussed when used against infectious diseases and as cancer vaccines. We propose a move away from replication-defective vaccines towards more robust replication-competent or single-cycle vaccines.

A rapid generation of adenovirus vector with a genetic modification in hexon protein.

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Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

2012-02-10

The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

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Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

2004-08-01

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

Combined adenovirus-mediated artificial microRNAs targeting mfgl2, mFas, and mTNFR1 protect against fulminant hepatic failure in mice.

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Dong Xi

Full Text Available Hepatitis B virus (HBV-related acute-on-chronic liver failure (ACLF has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2, FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA targeting mouse fgl2 (mfgl2 or both mFas and mTNFR1 on murine hepatitis virus (MHV-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4% mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2% mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7% mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

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Oki, Hiroshi; Yazawa, Takuya; Baba, Yasuko; Kanegae, Yumi; Sato, Hanako; Sakamoto, Seiko; Goto, Takahisa; Saito, Izumu; Kurahashi, Kiyoyasu

2017-07-01

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 10 9 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Complete replication-competent adenovirus 11p vectors with E1 or E3 insertions show improved heat stability

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Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se [Department of Clinical Microbiology and Virology, Umeå University, SE-901 85 Umeå (Sweden); Wu, Haidong, E-mail: haidong.wu@umu.se [Department of Clinical Microbiology and Virology, Umeå University, SE-901 85 Umeå (Sweden); Hultenby, Kjell, E-mail: kjell.hultenby@ki.se [Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institute, SE-14186 Stockholm (Sweden); Silver, Jim, E-mail: jim.silver@umu.se [Department of Clinical Microbiology and Virology, Umeå University, SE-901 85 Umeå (Sweden)

2016-10-15

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt;of the three, RCAd11pE3 was the most tolerant to heat treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47 °C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability. -- Highlights: •Replicating adenovirus 11p GFP vectors at the E1 or E3 region were generated. •RCAd11pE3 and RCAd11pE1 vectors manifested significantly improved heat stability. •RCAd11pE3 and RCAd11pE1 showed more full viral particles than Ad11pwt after heating. •We demonstrated that both genome size and the insertion site affect virion stability.

Complete replication-competent adenovirus 11p vectors with E1 or E3 insertions show improved heat stability

International Nuclear Information System (INIS)

Mei, Ya-Fang; Wu, Haidong; Hultenby, Kjell; Silver, Jim

2016-01-01

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt;of the three, RCAd11pE3 was the most tolerant to heat treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47 °C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability. -- Highlights: •Replicating adenovirus 11p GFP vectors at the E1 or E3 region were generated. •RCAd11pE3 and RCAd11pE1 vectors manifested significantly improved heat stability. •RCAd11pE3 and RCAd11pE1 showed more full viral particles than Ad11pwt after heating. •We demonstrated that both genome size and the insertion site affect virion stability.

Fowl adenovirus serotype 9 vectored vaccine for protection of avian influenza virus

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A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization.

Science.gov (United States)

Maxfield, Lori F; Abbink, Peter; Stephenson, Kathryn E; Borducchi, Erica N; Ng'ang'a, David; Kirilova, Marinela M; Paulino, Noelix; Boyd, Michael; Shabram, Paul; Ruan, Qian; Patel, Mayank; Barouch, Dan H

2015-11-01

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors. Copyright © 2015, Maxfield et al.

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

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Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

2017-08-15

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Science.gov (United States)

Carinhas, Nuno; Pais, Daniel A M; Koshkin, Alexey; Fernandes, Paulo; Coroadinha, Ana S; Carrondo, Manuel J T; Alves, Paula M; Teixeira, Ana P

2016-03-23

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

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Hiroyuki Mizuguchi

2011-07-01

Full Text Available The major limitation of the clinical use of replication-incompetent adenovirus (Ad vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN, following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs. In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88 and toll-like receptor 9 (TLR9 play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs, which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.

Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

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Unzu, Carmen; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo

2014-01-01

The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors. PMID:24465560

Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus.

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Carmen Unzu

Full Text Available The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.

Cancer-Targeted Oncolytic Adenoviruses for Modulation of the Immune System.

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Cerullo, Vincenzo; Capasso, Cristian; Vaha-Koskela, Markus; Hemminki, Otto; Hemminki, Akseli

2018-01-01

Adenovirus is one of the most commonly used vectors for gene therapy and it is the first approved virus-derived drug for treatment of cancer. As an oncolytic agent, it can induce lysis of infected cells, but it can also engage the immune system, promoting activation and maturation of antigen- presenting cells (APCs). In essence, oncolysis combined with the associated immunostimulatory actions result in a "personalized in situ vaccine" for each patient. In order to take full advantage of these features, we should try to understand how adenovirus interacts with the immune system, what are the receptors involved in triggering subsequent signals and which kind of responses they elicit. Tackling these questions will give us further insight in how to manipulate adenovirus-mediated immune responses for enhancement of anti-tumor efficacy. In this review, we first highlight how oncolytic adenovirus interacts with the innate immune system and its receptors such as Toll-like receptors, nucleotide-binding and oligomerization domain (NOD)- like receptors and other immune sensors. Then we describe the effect of these interactions on the adaptive immune system and its cells, especially B and T lymphocytes. Finally, we summarize the most significant preclinical and clinical results in the field of gene therapy where researchers have engineered adenovirus to manipulate the host immune system by expressing cytokines and signalingmediators. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon

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Roy, Soumitra; Shirley, Pamela S.; McClelland, Alan; Kaleko, Michael

1998-01-01

Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5. PMID:9658137

Development of an immunotherapeutic adenovirus targeting hormone-independent prostate cancer

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Kim JS

2013-11-01

Full Text Available Jae Sik Kim,1 Sang Don Lee,2 Sang Jin Lee,3 Moon Kee Chung21Department of Urology, The Catholic University of Korea Incheon St Mary's Hospital, Incheon, 2Pusan National University Yangsan Hospital and Research Institute for Convergence of Biomedical Science and Technology, Yangsan, 3Genitourinary Cancer Branch, National Cancer Center, Goyang, KoreaBackground: To develop a targeting therapy for hormone-independent prostate cancer, we constructed and characterized conditionally replicating oncolytic adenovirus (Ad equipped with mRFP(monomeric red fluorescence protein/ttk (modified herpes simplex virus thymidine kinase This construct was then further modified to express both mRFP/ttk and a soluble form of cytokine FLT3L (fms-related tyrosine kinase 3 ligand simultaneously.Methods: To construct the recombinant oncolytic adenovirus, E1a and E4 genes, which are necessary for adenovirus replication, were controlled by the prostate-specific enhancer sequence (PSES targeting prostate cancer cells expressing prostate-specific antigen (PSA and prostate-specific membrane antigen (PSMA. Simultaneously, it expressed the mRFP/ttk fusion protein in order to be able to elicit the cytotoxic effect.Results: The Ad5/35PSES.mRFP/ttk chimeric recombinant adenovirus was generated successfully. When replication of Ad5/35PSES.mRFP/ttk was evaluated in prostate cancer cell lines under fluorescence microscopy, red fluorescence intensity increased more in LNCaP cells, suggesting that the mRFP/ttk fusion protein was folded functionally. In addition, the replication assay including wild-type adenovirus as a positive control showed that PSES-positive cells (LNCaP and CWR22rv permitted virus replication but not PSES-negative cells (DU145 and PC3. Next, we evaluated the killing activity of this recombinant adenovirus. The Ad5/35PSES.mRFP/ttk killed LNCaP and CWR22rv more effectively. Unlike PSES-positive cells, DU145 and PC3 were resistant to killing by this recombinant

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

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Carey, John B; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V S; Draper, Simon J; Moore, Anne C

2014-08-21

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP1₄₂, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP1₄₂ also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP1₄₂ using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

Science.gov (United States)

Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

2014-01-01

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. PMID:25142082

Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts

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Jing Li Huang

2016-09-01

Full Text Available Oncolytic adenoviruses (OAds are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor, interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts.

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Huang, Jing Li; LaRocca, Christopher J; Yamamoto, Masato

2016-09-19

Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

Adenovirus dodecahedron, as a drug delivery vector.

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Monika Zochowska

Full Text Available BACKGROUND: Bleomycin (BLM is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad dodecahedron base (DB is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. PRINCIPAL FINDINGS: Dodecahedron (Dd structure is preserved at up to about 50 degrees C at pH 7-8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37 degrees C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. CONCLUSIONS/SIGNIFICANCE: Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.

Adenovirus-Vectored Vaccine as a Rapid-Response Tool Against Avian Influenza Pandemic

International Nuclear Information System (INIS)

Van Kampen, K. R.; Tang, D. C.

2007-01-01

Influenza viruses in nature undergo genetic mutation and reassortment. Three pandemics of avian influenza in man were recorded in the twentieth century. Highly pathogenic avian influenza (HPAI) viruses currently in circulation pose a threat for another world-wide pandemic, if they become transmissible from man to man. Manufacturing protective vaccines using current egg-based technology is often difficult due to the virulence of the virus and its adverse effects on the embryonating egg substrate. New technologies allow the creation of safe and protective pandemic influenza vaccines without the need for egg based substrates. These technologies allow new vaccines to be created in less than one month. Manufacturing is in tissue culture, not eggs. Vaccine can be administered to man non-invasively, without adjuvants, eliciting a rapid and protective immune response. Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad5)-derived vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 and H5N2 HPAI virus challenges. Mass-administration of this bird flu vaccine can be streamlined with available robotic in ovo injectors. Vaccination using this vaccine could protect the the largest host reservoir (chickens) and greatly reduce the exposure of man to avian influenza. In addition, Ad5-vectored vaccines can be produced rapidly and the safety margin of a non-replicating vector is superior to that of a replicating counterpart. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural AI virus infections. In addition to mass immunization of poultry, both animals and humans have been effectively immunized by intranasal administration of Ad5-vectored influenza vaccines without any appreciable side effects, even in mice and human volunteers with

Mucosal vaccination by adenoviruses displaying reovirus sigma 1

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Weaver, Eric A. [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Camacho, Zenaido T. [Department of Cell Biology, Department of Natural Sciences, Western New Mexico University, Silver City, NM 88062 (United States); Hillestad, Matthew L. [Nephrology Training Program, Mayo Clinic, Rochester, MN 55902 (United States); Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J. [Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902 (United States); Mercier, George T. [Department of Physics, University of Houston, Houston, TX 77004 (United States); Barry, Michael A., E-mail: mab@mayo.edu [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Department of Immunology and Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902 (United States)

2015-08-15

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

Mucosal vaccination by adenoviruses displaying reovirus sigma 1

International Nuclear Information System (INIS)

Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.; Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J.; Mercier, George T.; Barry, Michael A.

2015-01-01

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

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Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

2016-01-01

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

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Tomono, Takumi [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Kajita, Masahiro [Laboratory of Molecular Pharmaceutics and Technology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Yano, Kentaro [Laboratory of Biopharmaceutics, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Ogihara, Takuo, E-mail: togihara@takasaki-u.ac.jp [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan)

2016-08-05

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.

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Wenli Zhang

Full Text Available We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR and linear amplification-mediated PCR (LAM-PCR. Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

OpenAIRE

Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

2014-01-01

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delive...

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

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Guzman, Efrain; Taylor, Geraldine; Hope, Jayne; Herbert, Rebecca; Cubillos-Zapata, Carolina; Charleston, Bryan

2016-10-01

Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

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Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever.

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Sun, Yuan; Li, Hong-Yu; Tian, Da-Yong; Han, Qiu-Ying; Zhang, Xin; Li, Na; Qiu, Hua-Ji

2011-10-26

Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of replication-deficient adenovirus malaria vaccines.

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Hollingdale, Michael R; Sedegah, Martha; Limbach, Keith

2017-03-01

Malaria remains a major threat to endemic populations and travelers, including military personnel to these areas. A malaria vaccine is feasible, as radiation attenuated sporozoites induce nearly 100% efficacy. Areas covered: This review covers current malaria clinical trials using adenoviruses and pre-clinical research. Heterologous prime-boost regimens, including replication-deficient human adenovirus 5 (HuAd5) carrying malaria antigens, are efficacious. However, efficacy appears to be adversely affected by pre-existing anti-HuAd5 antibodies. Current strategies focus on replacing HuAd5 with rarer human adenoviruses or adenoviruses isolated from non-human primates (NHPs). The chimpanzee adenovirus ChAd63 is undergoing evaluation in clinical trials including infants in malaria-endemic areas. Key antigens have been identified and are being used alone, in combination, or with protein subunit vaccines. Gorilla adenoviruses carrying malaria antigens are also currently being evaluated in preclinical models. These replacement adenovirus vectors will be successfully used to develop vaccines against malaria, as well as other infectious diseases. Expert commentary: Simplified prime-boost single shot regimens, dry-coated live vector vaccines or silicon microneedle arrays could be developed for malaria or other vaccines. Replacement vectors with similar or superior immunogenicity have rapidly advanced, and several are now in extensive Phase 2 and beyond in malaria as well as other diseases, notably Ebola.

Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model

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Simão, Daniel; Pinto, Catarina; Fernandes, Paulo; Peddie, Christopher J.; Piersanti, Stefania; Collinson, Lucy M.; Salinas, Sara; Saggio, Isabella; Schiavo, Giampietro; Kremer, Eric J.; Brito, Catarina; Alves, Paula M.

2017-01-01

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in pre-clinical tests. For clinical translation, in-depth pre-clinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, while human adenovirus type 5 (HAdV5) showed increased tropism towards glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity. PMID:26181626

A recombinant E1-deleted porcine adenovirus-3 as an expression vector

International Nuclear Information System (INIS)

Zakhartchouk, Alexander; Zhou Yan; Tikoo, Suresh Kumar

2003-01-01

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B large coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B large of PAV-3 and also complemented PAV214 (E1A+E1B small deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B large coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B small + E1B large ) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B large was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

OpenAIRE

Warimwe, GM; Lorenzo, G; Lopez-Gil, E; Reyes-Sandoval, A; Cottingham, MG; Spencer, AJ; Collins, KA; Dicks, MD; Milicic, A; Lall, A; Furze, J; Turner, AV; Hill, AV; Brun, A; Gilbert, SC

2013-01-01

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibod...

Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats

International Nuclear Information System (INIS)

Liu Chunjie; Wang Dewen; Zhang Zhaoshan; Gao Yabing; Xiong Chengqi; Long Jianyin; Wang Huixin; Peng Ruiyun; Cui Xuemei

2001-01-01

Objective: To observed the efficiency of gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats. Methods: TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results: At day 1.5 after TGFβ1 sense and antisense gene transfer, PCR amplification using neo gene-specific primer from lung tissue DNA was all positive. After day 5.5, 67% (2/3) of lung tissue DNA was positive. RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo-antiTGFβ1 gene decreased. Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo-AntiTGFβ1 lipid complexes it raised remarkably (P 9 pfu/ml were instilled into bronchus at 0.5 ml per rat. After day 2 day 6, the lung tissues of all six rats (three per each group )expressed the transfected luciferase gene by luminometer. Conclusion: Cationic lipid-mediated TGFβ1 antisense gene therapy was a simple and easy method. It can slow down the course of pathogenesis of lung fibrosis. Replication-deficient recombinant adenovirus-mediated gene therapy of lung diseases is a good and efficient method

Use of adenoviral vectors as veterinary vaccines.

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Ferreira, T B; Alves, P M; Aunins, J G; Carrondo, M J T

2005-10-01

Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

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Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

2016-01-01

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

Neogenesis and proliferation of β-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

International Nuclear Information System (INIS)

Tokui, Yae; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro

2006-01-01

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of β-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a β-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of β-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of β-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted β-cell differentiation (mainly from duct cells) and proliferation of pre-existing β-cells, resulting in an increase of the β-cell mass that improved glucose tolerance in diabetic mice

Intraductal delivery of adenoviruses targets pancreatic tumors in transgenic Ela-myc mice and orthotopic xenografts.

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José, Anabel; Sobrevals, Luciano; Miguel Camacho-Sánchez, Juan; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina

2013-01-01

Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p less than 0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.

Future prospects for the development of cost-effective Adenovirus vaccines

DEFF Research Database (Denmark)

Fougeroux, Cyrielle; Holst, Peter J

2017-01-01

-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient...... as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes...

Protective immunity against tularemia provided by an adenovirus-vectored vaccine expressing Tul4 of Francisella tularensis.

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Kaur, Ravinder; Chen, Shan; Arévalo, Maria T; Xu, Qingfu; Chen, Yanping; Zeng, Mingtao

2012-03-01

Francisella tularensis, a category A bioterrorism agent, is a highly infectious organism that is passed on via skin contact and inhalation routes. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication-incompetent adenovirus, Ad/opt-Tul4, carrying a codon-optimized gene for expression of a membrane protein, Tul4, of F. tularensis LVS. Its ability to protect against lethal challenge and its immunogenicity were evaluated in a murine model. An intramuscular injection of a single dose (1 × 10(7) PFU) of Ad/opt-Tul4 elicited a robust Tul4-specific antibody response. Assays suggest a Th1-driven response. A single dose elicited 20% protection against challenge with 100 × 50% lethal dose (LD(50)) F. tularensis LVS; two additional booster shots resulted in 60% protection. In comparison, three doses of 5 μg recombinant Tul4 protein did not elicit significant protection against challenge. Therefore, the Ad/opt-Tul4 vaccine was more effective than the protein vaccine, and protection was dose dependent. Compared to LVS, the protection rate is lower, but an adenovirus-vectored vaccine may be more attractive due to its enhanced safety profile and mucosal route of delivery. Furthermore, simple genetic modification of the vaccine may potentially produce antibodies protective against a fully virulent strain of F. tularensis. Our data support the development and further research of an adenovirus-vectored vaccine against Tul4 of F. tularensis LVS.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

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Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

Crystal structure of the fibre head domain of the Atadenovirus Snake Adenovirus 1.

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Abhimanyu K Singh

Full Text Available Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1 fibre head using the multi-wavelength anomalous dispersion (MAD method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest.

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

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Andrew H. Baker

2010-10-01

Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

Science.gov (United States)

Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

2015-12-28

Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. Copyright © 2015 Elsevier B.V. All rights reserved.

MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

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Erkko Ylösmäki

Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

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Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

2007-04-01

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Adenovirus-vectored Ebola vaccines.

Science.gov (United States)

Gilbert, Sarah C

2015-01-01

The 2014 outbreak of Ebola virus disease in West Africa has highlighted the need for the availability of effective vaccines against outbreak pathogens that are suitable for use in frontline workers who risk their own health in the course of caring for those with the disease, and also for members of the community in the affected area. Along with effective contact tracing and quarantine, use of a vaccine as soon as an outbreak is identified could greatly facilitate rapid control and prevent the outbreak from spreading. This review describes the progress that has been made in producing and testing adenovirus-based Ebola vaccines in both pre-clinical and clinical studies, and considers the likely future use of these vaccines.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

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Alejo, Diana M; Moraes, Mauro P; Liao, Xiaofen; Dias, Camila C; Tulman, Edan R; Diaz-San Segundo, Fayna; Rood, Debra; Grubman, Marvin J; Silbart, Lawrence K

2013-04-26

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry. Copyright © 2013 Elsevier Ltd. All rights reserved.

HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells

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Chang Li

2018-06-01

Full Text Available We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR. The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ null (NSG mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr peptides (AcrII4 and AcrII2 capable of binding to the CRISPR/Cas9 complex (HDAd-Acr. CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 56.

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Duffy, Margaret R; Alonso-Padilla, Julio; John, Lijo; Chandra, Naresh; Khan, Selina; Ballmann, Monika Z; Lipiec, Agnieszka; Heemskerk, Evert; Custers, Jerome; Arnberg, Niklas; Havenga, Menzo; Baker, Andrew H; Lemckert, Angelique

2018-01-01

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

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El Andaloussi Samir

2011-05-01

Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

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Stefania Piersanti

Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

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Daniel M Appledorn

2010-03-01

Full Text Available Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+ and CD8(- T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

Cryo-EM structure of human adenovirus D26 reveals the conservation of structural organization among human adenoviruses.

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Yu, Xiaodi; Veesler, David; Campbell, Melody G; Barry, Mary E; Asturias, Francisco J; Barry, Michael A; Reddy, Vijay S

2017-05-01

Human adenoviruses (HAdVs) cause acute respiratory, ocular, and gastroenteric diseases and are also frequently used as gene and vaccine delivery vectors. Unlike the archetype human adenovirus C5 (HAdV-C5), human adenovirus D26 (HAdV-D26) belongs to species-D HAdVs, which target different cellular receptors, and is differentially recognized by immune surveillance mechanisms. HAdV-D26 is being championed as a lower seroprevalent vaccine and oncolytic vector in preclinical and human clinical studies. To understand the molecular basis for their distinct biological properties and independently validate the structures of minor proteins, we determined the first structure of species-D HAdV at 3.7 Å resolution by cryo-electron microscopy. All the hexon hypervariable regions (HVRs), including HVR1, have been identified and exhibit a distinct organization compared to those of HAdV-C5. Despite the differences in the arrangement of helices in the coiled-coil structures, protein IX molecules form a continuous hexagonal network on the capsid exterior. In addition to the structurally conserved region (3 to 300) of IIIa, we identified an extra helical domain comprising residues 314 to 390 that further stabilizes the vertex region. Multiple (two to three) copies of the cleaved amino-terminal fragment of protein VI (pVIn) are observed in each hexon cavity, suggesting that there could be ≥480 copies of VI present in HAdV-D26. In addition, a localized asymmetric reconstruction of the vertex region provides new details of the three-pronged "claw hold" of the trimeric fiber and its interactions with the penton base. These observations resolve the previous conflicting assignments of the minor proteins and suggest the likely conservation of their organization across different HAdVs.

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors

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Catherine M. Crosby

2017-02-01

Full Text Available Most adenovirus (Ad vectors are E1 gene deleted replication defective (RD-Ad vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

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Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals.

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Fuchs, Jonathan D; Bart, Pierre-Alexandre; Frahm, Nicole; Morgan, Cecilia; Gilbert, Peter B; Kochar, Nidhi; DeRosa, Stephen C; Tomaras, Georgia D; Wagner, Theresa M; Baden, Lindsey R; Koblin, Beryl A; Rouphael, Nadine G; Kalams, Spyros A; Keefer, Michael C; Goepfert, Paul A; Sobieszczyk, Magdalena E; Mayer, Kenneth H; Swann, Edith; Liao, Hua-Xin; Haynes, Barton F; Graham, Barney S; McElrath, M Juliana

2015-05-01

Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted.

Adenovirus-derived vectors for prostate cancer gene therapy

Czech Academy of Sciences Publication Activity Database

de Vrij, J.; Willemsen, R. A.; Lindholm, L.; Hoeben, R. C.; Bangma, Ch. H.; Barber, Ch.; Behr, J.-P.; Briggs, S.; Carlisle, R.; Cheng, W.-S.; Dautzenberg, I. J. C.; de Ridder, C.; Dzojic, H.; Erbacher, P.; Essand, M.; Fisher, K.; Frazier, A.; Georgopoulos, L. J.; Jennings, I.; Kochanek, S.; Koppers-Lalic, D.; Kraaij, R.; Kreppel, F.; Magnusson, M.; Maitland, N.; Neuberg, P.; Nugent, R.; Ogris, M.; Remy, J.-S.; Scaife, M.; Schenk, E.; Schooten, E.; Seymour, L.; Slade, M.; Szyjanowicz, P.; Totterman, T.; Uil, T. G.; Ulbrich, Karel; van der Weel, L.; van Weerden, W.; Wagner, E.; Zuber, G.

2010-01-01

Roč. 21, č. 7 (2010), s. 795-805 ISSN 1043-0342 EU Projects: European Commission(XE) 512087 - GIANT Keywords : adenovirus * gene delivery * prostate cancer Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.829, year: 2010

Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus.

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Zhi, Yan; Figueredo, Joanita; Kobinger, Gary P; Hagan, Heather; Calcedo, Roberto; Miller, James R; Gao, Guangping; Wilson, James M

2006-05-01

Replication-deficient human adenovirus type 5 (AdH5) vectors can induce strong transgene product-specific cellular and humoral responses. However, many adult humans have neutralizing antibodies (NAbs) against AdH5 as a result of natural infection with this virus. Therefore, a chimpanzee adenovirus C7 (AdC7) vector was developed to circumvent interference by preexisting immunity to AdH5. This study evaluated the impact of preexisting immunity to human adenovirus on the efficacy of adenovirus-based vaccines against the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). Efficacy was assessed after intramuscular injection of the vector into mice and was measured as the frequency of SARS-CoV-specific T cells and NAbs against SARS-CoV. Immunogenicity of the AdH5-based vaccine was significantly attenuated or completely abolished when the preexisting anti-AdH5 NAb titer was higher than 40. Because 27% of human serum samples from the United States tested so far have an anti-AdH5 NAb titer higher than 40, our results suggested that a significant percentage of humans with preexisting anti-AdH5 immunity would not be candidates for vaccination with an AdH5-based genetic vaccine. In contrast, preexisting anti-AdH5 NAbs have a minimal effect on the potency of the AdC7-based genetic vaccine. Taken together, our studies warrant the further development of AdC7 as a vaccine carrier for human trials.

In vitro radionuclide therapy and in vivo scintigraphic imaging of alpha fetoprotein producing hepatocellular carcinoma by targeted sodium iodide symporter gene expression

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Kim, Kwang Il; Lee, Yong Jin; Lee, Tae Sup; Song, Inho; Cheon, Gi Jeong; Lim, Sang Moo; Kang, Joo Hyun [Korea Institute of Radiological and Medical and Medical Sciences, Seoul (Korea, Republic of); Chung, June Key [Seoul National Univ. College of Medicine, Seoul (Korea, Republic of)

2012-03-15

This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha fetoprotein (AFP) producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter. The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP producing cells and in AFP nonproducing cells was investigated using {sup 125}I uptake assay and semi quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of {sup 131}I vitro clonogenic assay. In addition, tumor bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained. The expression of hNIS was efficiently demonstrated by {sup 125}I uptake assay in AFP producing cells, but not in AFP nonproducing cells. AFP producing HCC targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP producing cells caused more sensitivity to {sup 131}I than that in AFP nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging. An AFP producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP producing HCC specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system.

In vitro radionuclide therapy and in vivo scintigraphic imaging of alpha fetoprotein producing hepatocellular carcinoma by targeted sodium iodide symporter gene expression

International Nuclear Information System (INIS)

Kim, Kwang Il; Lee, Yong Jin; Lee, Tae Sup; Song, Inho; Cheon, Gi Jeong; Lim, Sang Moo; Kang, Joo Hyun; Chung, June Key

2012-01-01

This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha fetoprotein (AFP) producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter. The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP producing cells and in AFP nonproducing cells was investigated using 125 I uptake assay and semi quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of 131 I vitro clonogenic assay. In addition, tumor bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained. The expression of hNIS was efficiently demonstrated by 125 I uptake assay in AFP producing cells, but not in AFP nonproducing cells. AFP producing HCC targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP producing cells caused more sensitivity to 131 I than that in AFP nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging. An AFP producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP producing HCC specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system

Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

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Akikazu Ishihara

2012-01-01

Full Text Available Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad, serotype 2 adeno-associated virus vectors (rAAV2, or self-complementary (sc AAV2 vectors carrying green fluorescent protein (GFP. Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

Adenovirus Detection by the cGAS/STING/TBK1 DNA Sensing Cascade

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Lam, Eric; Stein, Saskia

2014-01-01

Adenovirus (Ad) infection triggers a cell-specific antiviral response following exposure of viral DNA to the intracellular compartment. A variety of DNA sensors (DAI, AIM2, DDx41, RNA polymerase [Pol] III, and IFI16 [p204]) have been identified in recent years; however, the DNA sensor involved in detection of adenovirus has not been established. Cyclic GMP-AMP synthase (cGAS), a DNA sensor that produces a cyclic guanine-adenine dinucleotide (cGAMP) inducer of STING, has been examined to determine its role in generating an antiadenoviral response. Short hairpin RNA (shRNA) lentiviral vectors targeting TBK1, STING, and cGAS were established in murine MS1 endothelial and RAW 264.7 macrophage cell lines. Knockdown of TBK1, STING, and cGAS results in a dramatic reduction in the activation of the primary antiviral response marker phosphorylated interferon (IFN) response factor 3 (IRF3) following exposure to adenovirus. Furthermore, activation of secondary type I IFN signaling targets (ptyrSTAT1 and ptyrSTAT2 [ptyrSTAT1/2]) was also compromised. Consistent with compromised activation of primary and secondary response markers, transcriptional activation of IRF3-responsive genes (beta IFN [IFN-β], ISG15, ISG54) and secondary response transcripts were diminished in cells knocked down in cGAS, STING, or TBK1. These data establish cGAS as the dominant cytosolic DNA sensor responsible for detection of internalized adenovirus leading to induction of the type I interferon antiviral cascade. PMID:24198409

[Construction and expression of a recombinant adenovirus with LZP3].

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Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

2007-08-01

To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

PDGF-receptor beta-targeted adenovirus redirects gene transfer from hepatocytes to activated stellate cells

NARCIS (Netherlands)

Schoemaker, Marieke H.; Rots, Marianne G.; Beljaars, Leonie; Ypma, Arjen Y.; Jansen, Peter L. M.; Poelstra, Klaas; Moshage, Albert; Haisma, Hidde J.

2008-01-01

Chronic liver damage may lead to liver fibrosis. In this process, hepatic activated stellate cells are the key players. Thus, activated stellate cells are attractive targets for antifibrotic gene therapy. Recombinant, adenovirus is a promising vehicle for delivering therapeutic genes to liver cells.

Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

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Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

2006-08-01

Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

Science.gov (United States)

Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

2005-12-20

Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

Immunization with a Novel Human type 5 Adenovirus-Vectored Vaccine Expressing the Premembrane and Envelope Proteins of Zika Virus Provides Consistent and Sterilizing Protection in Multiple Immunocompetent and Immunocompromised Animal Models.

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Guo, Qiang; Chan, Jasper Fuk-Woo; Poon, Vincent Kwok-Man; Wu, Shipo; Chan, Chris Chung-Sing; Hou, Lihua; Yip, Cyril Chik-Yan; Ren, Changpeng; Cai, Jian-Piao; Zhao, Mengsu; Zhang, Anna Jinxia; Song, Xiaohong; Chan, Kwok-Hung; Wang, Busen; Kok, Kin-Hang; Wen, Yanbo; Yuen, Kwok-Yung; Chen, Wei

2018-03-29

Zika virus (ZIKV) infection may be associated with severe complications and disseminated via both vector-borne and non-vector-borne routes. Adenovirus-vectored vaccines represent a favorable controlling measure for the ZIKV epidemic as they have been shown to be safe, immunogenic, and rapidly generable for other emerging viral infections. Evaluations of two previously reported adenovirus-vectored ZIKV vaccines were performed using non-lethal animal models and/or non-epidemic ZIKV strain. We constructed and evaluated two human adenovirus-5-vectored vaccines containing the ZIKV premembrane-envelope(Ad5-Sig-prM-Env) and envelope(Ad5-Env) proteins, respectively, in multiple non-lethal and lethal animal models using epidemic ZIKV strains. Both vaccines elicited robust humoral and cellular immune responses in immunocompetent BALB/c mice. Dexamethasone-immunosuppressed mice vaccinated with either vaccine demonstrated robust and durable antibody responses and significantly lower blood/tissue viral loads than controls(Panimal models, Ad5-Sig-prM-Env-vaccinated mice had significantly(P<0.05) higher titers of anti-ZIKV-specific neutralizing antibody titers and lower(undetectable) viral loads than Ad5-Env-vaccinated mice. The close correlation between the neutralizing antibody titer and viral load helped to explain the better protective effect of Ad5-Sig-prM-Env than Ad5-Env. Anamnestic response was absent in Ad5-Sig-prM-Env-vaccinated A129 mice. Ad5-Sig-prM-Env provided sterilizing protection against ZIKV infection in mice.

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

Science.gov (United States)

Maler, Mareike D; Nielsen, Peter J; Stichling, Nicole; Cohen, Idan; Ruzsics, Zsolt; Wood, Connor; Engelhard, Peggy; Suomalainen, Maarit; Gyory, Ildiko; Huber, Michael; Müller-Quernheim, Joachim; Schamel, Wolfgang W A; Gordon, Siamon; Jakob, Thilo; Martin, Stefan F; Jahnen-Dechent, Willi; Greber, Urs F; Freudenberg, Marina A; Fejer, György

2017-08-01

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages

Epidermal growth factor receptor targeting of replication competent adenovirus enhances cytotoxicity in bladder cancer

NARCIS (Netherlands)

van der Poel, HG; Molenaar, B; van Beusechem, VW; Haisma, HJ; Rodriguez, R; Curiel, DT; Gerritsen, WR

Purpose: We evaluated the delivery and oncolytic potential of targeted replication competent adenoviruses in bladder cancer lines. Materials and Methods: Seven established human bladder cancer tumor lines (5637, SW800, TCCsup, J82, Scaber, T24 and 253J) were studied for the expression of integrins

A novel oncolytic adenovirus targeting Wnt signaling effectively inhibits cancer-stem like cell growth via metastasis, apoptosis and autophagy in HCC models.

Science.gov (United States)

Zhang, Jian; Lai, Weijie; Li, Qiang; Yu, Yang; Jin, Jin; Guo, Wan; Zhou, Xiumei; Liu, Xinyuan; Wang, Yigang

2017-09-16

Cancer stem cells (CSCs), which are highly differentiated and self-renewing, play an important role in the occurrence, therapeutic resistant and metastasis of hepatacellular carcinoma (HCC). Oncolytic adenoviruses have targeted killing effect on tumor cells, and are invoked as candidate drugs for cancer treatment. We designed a dual-regulated oncolytic adenovirus Ad.wnt-E1A(△24bp)-TSLC1 that targets Wnt and Rb signaling pathways respectively, and carries the tumor suppressor gene, TSLC1. Previous studies have demonstrated that oncolytic adenovirus mediated TSLC1can target liver cancer and exhibit significant cytotoxicity. However, whether Ad.wnt-E1A(△24bp)-TSLC1 can effectively eliminate liver CSCs remains to be explored. We first used the spheroid culture to enrich the liver CSCs-like cells, and detected the self-renewal capacity, differentiation, drug resistance and tumorigenicity. The results showed that Ad-wnt-E1A(△24bp)-TSLC1 could effectively lead to autophagic death. In addition, recombinant adenovirus effectively induced the apoptosis, inhibit metastasis of hepatic CSCs-like cells in vivo. Further animal experiments indicated that Ad-wnt-E1A(△24bp)-TSLC1could effectively inhibit the growth of transplanted tumor of hepatic CSCs and prolong the survival time of mice. Therefore, the novel oncolytic adenovirus Ad.wnt-E1A(△24bp)-TSLC1 has potential application as a therapeutic target for HCC stem cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production.

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Ferreira, Tiago B; Ferreira, Ana L; Carrondo, Manuel J T; Alves, Paula M

2005-11-01

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

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Limbach, Keith; Stefaniak, Maureen; Chen, Ping; Patterson, Noelle B; Liao, Grant; Weng, Shaojie; Krepkiy, Svetlana; Ekberg, Greg; Torano, Holly; Ettyreddy, Damodar; Gowda, Kalpana; Sonawane, Sharvari; Belmonte, Arnel; Abot, Esteban; Sedegah, Martha; Hollingdale, Michael R; Moormann, Ann; Vulule, John; Villasante, Eileen; Richie, Thomas L; Brough, Douglas E; Bruder, Joseph T

2017-07-03

A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Time-dependent biodistribution and transgene expression of a recombinant human adenovirus serotype 5-luciferase vector as a surrogate agent for rAd5-FMDV vaccines in cattle

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Replication-defective recombinant adenovirus 5 (rAd5) vectors carrying foot-and-mouth disease virus (FMDV) transgenes elicit a robust immune response to FMDV challenge in cattle; however vaccine function mechanisms are incompletely understood. Recent efforts addressing critical interactions of rAd5 ...

Mucosal vaccination with heterologous viral vectored vaccine targeting subdominant SIV accessory antigens strongly inhibits early viral replication

DEFF Research Database (Denmark)

Xu, Huanbin; Andersson, Anne-Marie Carola; Ragonnaud, Emeline

2017-01-01

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat...

Protection of non-human primates against rabies with an adenovirus recombinant vaccine

International Nuclear Information System (INIS)

Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

2014-01-01

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus

Protection of non-human primates against rabies with an adenovirus recombinant vaccine

Energy Technology Data Exchange (ETDEWEB)

Xiang, Z.Q. [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Greenberg, L. [Centers for Disease Control and Prevention, Atlanta, GA (United States); Ertl, H.C., E-mail: ertl@wistar.upenn.edu [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Rupprecht, C.E. [The Global Alliance for Rabies Control, Manhattan, KS (United States); Ross University School of Veterinary Medicine, Basseterre (Saint Kitts and Nevis)

2014-02-15

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

Species D human adenovirus type 9 exhibits better virus-spread ability for antitumor efficacy among alternative serotypes.

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Junji Uchino

Full Text Available Species C human adenovirus serotype 5 (HAdV-C5 is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR, HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.

Adeno-associated virus vectors can be efficiently produced without helper virus.

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Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

1998-07-01

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Chimpanzee Adenovirus Vector Ebola Vaccine.

Science.gov (United States)

Ledgerwood, Julie E; DeZure, Adam D; Stanley, Daphne A; Coates, Emily E; Novik, Laura; Enama, Mary E; Berkowitz, Nina M; Hu, Zonghui; Joshi, Gyan; Ploquin, Aurélie; Sitar, Sandra; Gordon, Ingelise J; Plummer, Sarah A; Holman, LaSonji A; Hendel, Cynthia S; Yamshchikov, Galina; Roman, Francois; Nicosia, Alfredo; Colloca, Stefano; Cortese, Riccardo; Bailer, Robert T; Schwartz, Richard M; Roederer, Mario; Mascola, John R; Koup, Richard A; Sullivan, Nancy J; Graham, Barney S

2017-03-09

The unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10 10 particle units or 2×10 11 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10 11 particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10 11 particle-unit dose than in the group that received the 2×10 10 particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×10 11 particle-unit dose than among those who received the 2×10 10 particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×10 11 particle-unit dose. Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At

Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

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Zhao Yarui

2011-01-01

Full Text Available Abstract Background Sphingomyelin synthase 2 (SMS2 contributes to de novo sphingomyelin (SM biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. Methods The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR and protein level examination (SMS activity assay. Result We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2 or GFP cDNA (AdV-GFP. On day six after intravenous infusion of 2 × 1011 particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p Conclusions Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.

Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

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Minglong Liu

Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein region II of Plasmodium vivax

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Simone C de Cassan

2015-07-01

Full Text Available Malaria vaccine development has largely focused on Plasmodium falciparum; however a reawakening to the importance of P. vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII with the human Duffy antigen receptor for chemokines (DARC, makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically-compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5, chimpanzee adenovirus serotype 63 (ChAd63 and modified vaccinia virus Ankara (MVA vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime, or in ‘mixed-modality’ adenovirus prime – protein-in-adjuvant boost regimes (using a recombinant protein PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants. Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII and have recently entered clinical trials which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans.

Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors.

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Yamashita, Toshiharu; Okura, Masae; Ishii-Osai, Yasue; Hida, Tokimasa

2016-10-01

Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP-A to -G, and a variant type (XP-V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP-A, -B, -C, -D, -F or -G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV-C at 5-20 J/m 2 was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP-E and XP-V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co-infection of Ad-XPA with Ad-XPE increased survival rate of XP-E cells after UV-C exposure. When XP-V cell strains, including one derived from a Japanese patient, were infected with Ad-XPV, exposed to UV-B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP-V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP-E and XP-V. © 2016 Japanese Dermatological Association.

[Adenovirus-mediated canine interferon-gamma expression and its antiviral activity against canine parvovirus].

Science.gov (United States)

Zhang, Kao; Jin, Huijun; Zhong, Fei; Li, Xiujin; Neng, Changai; Chen, Huihui; Li, Wenyan; Wen, Jiexia

2012-11-04

To construct recombinant adenovirus containing canine interferon-gamma (cIFN-gamma) gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells (MDCK). [Methods] The cIFN-gamma gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-gamma expression vector, from which the cIFN-gamma expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-gamma. The pAd-cIFN-gamma plasmid was linearized by digestion and transfected into human embryonic kidney (HEK) 293T cells to generate the replication-defective cIFN-gamma recombinant adenovirus (Ad-cIFN-gamma). To analyze its anti-canine parvovirus activity, the MDCK cells were pre-infected by Ad-cIFN-gamma recombinant adenovirus, and then infected by canine parvovirus. The antiviral activity of the Ad-cIFN-gamma recombinant adenovirus against parvovirus was analyzed. The recombinant adenovirus containing cIFN-gamma gene was constructed by the ligation method. The recombinant adenovirus could mediates recombinant cIFN-gamma secretory expression in MDCK cells. The Ad-cIFN-gamma recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus. These results indicate that the Ad-cIFN-gamma recombinant adenovirus has the potent antiviral activity against canine parvovirus. The Ad-cIFN-gamma recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus.

Poxvirus-vectored vaccines for rabies--a review.

Science.gov (United States)

Weyer, Jacqueline; Rupprecht, Charles E; Nel, Louis H

2009-11-27

Oral rabies vaccination of target reservoir species has proved to be one of the pillars of successful rabies elimination programs. The use of live attenuated rabies virus vaccines has been extensive but several limitations hamper its future use. A recombinant vaccinia-rabies vaccine has also been successfully used for the oral vaccination of several species. Nevertheless, its lack of efficacy in certain important rabies reservoirs and concerns on the use of this potent live virus as vaccine carrier (vector) impair the expansion of its use for new target species and new areas. Several attenuated and host-restricted poxvirus alternatives, which supposedly offer enhanced safety, have been investigated. Once again, efficacy in certain target species and innocuity through the oral route remain major limitations of these vaccines. Alternative recombinant vaccines using adenovirus as an antigen delivery vector have been extensively investigated and may provide an important addition to the currently available oral rabies vaccine repertoire, but are not the primary subject of this review.

Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

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Anton V Borovjagin

Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

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Richardson, Jason S; Yao, Michel K; Tran, Kaylie N; Croyle, Maria A; Strong, James E; Feldmann, Heinz; Kobinger, Gary P

2009-01-01

The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the

Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

Directory of Open Access Journals (Sweden)

Jason S Richardson

Full Text Available BACKGROUND: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP. The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. METHODOLOGY/PRINCIPAL FINDINGS: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP. Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. CONCLUSIONS/SIGNIFICANCE: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the

Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges

International Nuclear Information System (INIS)

Wang Danher; Hevey, Michael; Juompan, Laure Y.; Trubey, Charles M.; Raja, Nicholas U.; Deitz, Stephen B.; Woraratanadharm, Jan; Luo Min; Yu Hong; Swain, Benjamin M.; Moore, Kevin M.; Dong, John Y.

2006-01-01

The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10 7 pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV

Oncolytic Adenoviruses in Cancer Treatment

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Ramon Alemany

2014-02-01

Full Text Available The therapeutic use of viruses against cancer has been revived during the last two decades. Oncolytic viruses replicate and spread inside tumors, amplifying their cytotoxicity and simultaneously reversing the tumor immune suppression. Among different viruses, recombinant adenoviruses designed to replicate selectively in tumor cells have been clinically tested by intratumoral or systemic administration. Limited efficacy has been associated to poor tumor targeting, intratumoral spread, and virocentric immune responses. A deeper understanding of these three barriers will be required to design more effective oncolytic adenoviruses that, alone or combined with chemotherapy or immunotherapy, may become tools for oncologists.

The structural basis for the integrity of adenovirus Ad3 dodecahedron.

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Ewa Szolajska

Full Text Available During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3 synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47, as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

National Research Council Canada - National Science Library

Huang, Shuang

2004-01-01

.... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

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Iizuka, Shunsuke; Sakurai, Fuminori; Tachibana, Masashi; Ohashi, Kazuo; Mizuguchi, Hiroyuki

2017-09-15

Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

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Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

2004-11-01

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

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Osada, Takuya; Morse, Michael A; Hobeika, Amy; Lyerly, H Kim

2012-06-01

Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

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Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

2013-02-28

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

Activated recombinant adenovirus proteinases

Science.gov (United States)

Anderson, Carl W.; Mangel, Walter F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Inhibitory effect of Survivin promoter-regulated oncolytic adenovirus carrying P53 gene against gallbladder cancer.

Science.gov (United States)

Liu, Chen; Sun, Bin; An, Ni; Tan, Weifeng; Cao, Lu; Luo, Xiangji; Yu, Yong; Feng, Feiling; Li, Bin; Wu, Mengchao; Su, Changqing; Jiang, Xiaoqing

2011-12-01

Gene therapy has become an important strategy for treatment of malignancies, but problems remains concerning the low gene transferring efficiency, poor transgene expression and limited targeting specific tumors, which have greatly hampered the clinical application of tumor gene therapy. Gallbladder cancer is characterized by rapid progress, poor prognosis, and aberrantly high expression of Survivin. In the present study, we used a human tumor-specific Survivin promoter-regulated oncolytic adenovirus vector carrying P53 gene, whose anti-cancer effect has been widely confirmed, to construct a wide spectrum, specific, safe, effective gene-viral therapy system, AdSurp-P53. Examining expression of enhanced green fluorecent protein (EGFP), E1A and the target gene P53 in the oncolytic adenovirus system validated that Survivin promoter-regulated oncolytic adenovirus had high proliferation activity and high P53 expression in Survivin-positive gallbladder cancer cells. Our in vitro cytotoxicity experiment demonstrated that AdSurp-P53 possessed a stronger cytotoxic effect against gallbladder cancer cells and hepatic cancer cells. The survival rate of EH-GB1 cells was lower than 40% after infection of AdSurp-P53 at multiplicity of infection (MOI) = 1 pfu/cell, while the rate was higher than 90% after infection of Ad-P53 at the same MOI, demonstrating that AdSurp-P53 has a potent cytotoxicity against EH-GB1 cells. The tumor growth was greatly inhibited in nude mice bearing EH-GB1 xenografts when the total dose of AdSurp-P53 was 1 × 10(9) pfu, and terminal dUTP nick end-labeling (TUNEL) revealed that the apoptotic rate of cancer cells was (33.4 ± 8.4)%. This oncolytic adenovirus system overcomes the long-standing shortcomings of gene therapy: poor transgene expression and targeting of only specific tumors, with its therapeutic effect better than the traditional Ad-P53 therapy regimen already on market; our system might be used for patients with advanced gallbladder cancer and

A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

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Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

2015-09-01

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

Development of a nasal adenovirus-based vaccine: Effect of concentration and formulation on adenovirus stability and infectious titer during actuation from two delivery devices.

Science.gov (United States)

Renteria, Sandra S; Clemens, Courtney C; Croyle, Maria A

2010-02-25

A nasal adenovirus-based vaccine is under development. To determine if aggregation occurs during vaccination, infectious titer (limiting dilution) and capsid integrity (dynamic light scattering) were assessed after extrusion of a model vector from two intranasal delivery devices. Preparations of 2.5x10(12) and 1.25x10(11) virus particles (vp)/ml were studied. Virus aggregated ( approximately 10%) in the multi-dose vessel. Virus titer dropped by one log. Virus in the unit-dose device aggregated ( approximately 1%). Titer remained unchanged. Aggregation was concentration dependent. Formulations prevented aggregation during actuation, freeze-thaw and long-term storage. The device, formulation and dose may significantly influence aggregation and potency of any nasal adenovirus 5-based vaccine. Copyright 2009 Elsevier Ltd. All rights reserved.

Mechanism of ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells.

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Cheng Cheng

2007-02-01

Full Text Available Recombinant adenoviral (rAd vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5 vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs was independent of the coxsackievirus and adenovirus receptor (CAR, its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines.

Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement.

Science.gov (United States)

Kim, Sung Jin; Kim, Ju Hyun; Yang, Bitna; Jeong, Jin-Sook; Lee, Seong-Wook

2017-02-01

Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

Science.gov (United States)

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel J; Kalle, Wouter H J

2005-06-01

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Interferon induction by adenoviruses

Energy Technology Data Exchange (ETDEWEB)

Beladi, I; Bakay, M; Pusztai, R; Mucsi, I; Tarodi, B [University Medical School, Szeged (Hungary). Inst. of Microbiology

1979-02-01

All human, simian, bovine and avian adenovirus types tested so far and the canine hepatitis virus induce interferon production in chick cells. This finding indicated this property to be characteristic for viruses belonging to the adenovirus group. Trypsin treatment, which had no effect upon the infectivity, diminished or eliminated the interferon-inducing abilities of crude adenoviruses, and thus the need for a trypsin-sensitive protein in interferon induction was suggested. T antigen and interferon were formed simultaneously in chick embryo fibroblast cells infected with human adenovirus type 12, and there-fore the adenovirus-specific T antigen was resitant to the action of endogenous interferon synthetized by the same cells. In chicks inoculated with human types, the appearance of interferon was biphasic: an 'early' and a 'late' interferon could be demonstrated with maximum titre 4 and 10 hr, respectively, after virus infection. In chicks infected with adenoviruses, first interferon production and then a decreased primary immune response to sheep red blood cells was observed. It was assumed that in adenovirus-infected chicks the interferon produced by viral stimulus resulted in a transient immunosuppression.

A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism

International Nuclear Information System (INIS)

Takayama, Koichi; Reynolds, Paul N.; Short, Joshua J.; Kawakami, Yosuke; Adachi, Yasuo; Glasgow, Joel N.; Rots, Marianne G.; Krasnykh, Victor; Douglas, Joanne T.; Curiel, David T.

2003-01-01

The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion

Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

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Lucile Tran

Full Text Available Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT imaging of gene expression to determine whether local virus administration (direct injection in the kidney could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting.

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines.

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See, Raymond H; Petric, Martin; Lawrence, David J; Mok, Catherine P Y; Rowe, Thomas; Zitzow, Lois A; Karunakaran, Karuna P; Voss, Thomas G; Brunham, Robert C; Gauldie, Jack; Finlay, B Brett; Roper, Rachel L

2008-09-01

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

Co-factor activated recombinant adenovirus proteinases

Science.gov (United States)

Anderson, Carl W.; Mangel, Walter F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

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Guo, H.H.; Yu, C.C.; Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China); Ma, X.J. [Ningxia Medical Autonomous Region of the First People' s Hospital, Department of Orthopedic Surgery, Yinchuan (China); Yang, X.C.; Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China)

2013-10-02

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

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Anil Babu Korrapati

Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

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Philip Ng

2010-09-01

Full Text Available Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application.

Protection of Non-Human Primates against Rabies with an Adenovirus Recombinant Vaccine

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Xiang, Z.Q.; Greenberg, L.; Ertl, H. C.; Rupprecht, C.E.

2014-01-01

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. PMID:24503087

Eradication of melanoma in vitro and in vivo via targeting with a Killer-Red-containing telomerase-dependent adenovirus.

Science.gov (United States)

Takehara, Kiyoto; Yano, Shuya; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Narii, Nobuhiro; Mizuguchi, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

2017-08-18

Melanoma is a highly recalcitrant cancer and transformative therapy is necessary for the cure of this disease. We recently developed a telomerase-dependent adenovirus containing the fluorescent protein Killer-Red. In the present report, we first determined the efficacy of Killer-Red adenovirus combined with laser irradiation on human melanoma cell lines in vitro. Cell viability of human melanoma cells was reduced in a dose-dependent and irradiation-time-dependent manner. We used an intradermal xenografted melanoma model in nude mice to determine efficacy of the Killer-Red adenovirus. Intratumoral injection of Killer-Red adenovirus, combined with laser irradiation, eradicated the melanoma indicating the potential of a new paradigm of cancer therapy.

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

International Nuclear Information System (INIS)

Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

2004-01-01

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice

OpenAIRE

Crettaz, J. (Julien); Berraondo, P. (Pedro); Mauleon, I. (Itsaso); Ochoa, L. (Laura); Shankar, V. (Vijay); Barajas, M. (Miguel); Rooijen, N. (Nico) van; Kochanek, S. (Stefan); Qian, C. (Cheng); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Gonzalez-Aseguinolaza, G. (Gloria)

2006-01-01

Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH in...

Mouse adenovirus type 1 infection of macrophages

NARCIS (Netherlands)

Ashley, S.L.; Welton, A.R.; Harwood, K.M.; Rooijen, van N.; Spindler, K.R.

2009-01-01

Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus.

Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

Science.gov (United States)

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio

2012-10-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

Viral Vectors for Use in the Development of Biodefense Vaccines

National Research Council Canada - National Science Library

Lee, John S; Hadjipanayis, Angela G; Parker, Michael D

2005-01-01

... agents of bioterrorism or biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine-vectors has enabled researchers to develop effective means for countering the threat of bioterrorism and biowarfare. An overview of the different viral vectors and the threats they counter will be discussed.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

Energy Technology Data Exchange (ETDEWEB)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds.

Directory of Open Access Journals (Sweden)

Michael Behr

Full Text Available Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK. This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

Science.gov (United States)

Behr, Michael; Kaufmann, Johanna K.; Ketzer, Patrick; Engelhardt, Sarah; Mück-Häusl, Martin; Okun, Pamela M.; Petersen, Gabriele; Neipel, Frank; Hassel, Jessica C.; Ehrhardt, Anja; Enk, Alexander H.; Nettelbeck, Dirk M.

2014-01-01

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis. PMID:24760010

Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

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Shen-shen ZHI

2011-02-01

Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

Vector-transmitted disease vaccines: targeting salivary proteins in transmission (SPIT).

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McDowell, Mary Ann

2015-08-01

More than half the population of the world is at risk for morbidity and mortality from vector-transmitted diseases, and emerging vector-transmitted infections are threatening new populations. Rising insecticide resistance and lack of efficacious vaccines highlight the need for novel control measures. One such approach is targeting the vector-host interface by incorporating vector salivary proteins in anti-pathogen vaccines. Debate remains about whether vector saliva exposure exacerbates or protects against more severe clinical manifestations, induces immunity through natural exposure or extends to all vector species and associated pathogens. Nevertheless, exploiting this unique biology holds promise as a viable strategy for the development of vaccines against vector-transmitted diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination.

Science.gov (United States)

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

Directory of Open Access Journals (Sweden)

Julien Revaud

2018-01-01

Full Text Available To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

Science.gov (United States)

Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

2016-01-01

The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

International Nuclear Information System (INIS)

Ugai, Hideyo; Yamasaki, Takahito; Hirose, Megumi; Inabe, Kumiko; Kujime, Yukari; Terashima, Miho; Liu, Bingbing; Tang, Hong; Zhao, Mujun; Murata, Takehide; Kimura, Makoto; Pan, Jianzhi; Obata, Yuichi; Hamada, Hirofumi; Yokoyama, Kazunari K.

2005-01-01

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 deg C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10 10 pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5

Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

Energy Technology Data Exchange (ETDEWEB)

Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

2009-02-15

expression by polymerase chain reaction in adenovirus vector-infected cardiac myocytes was significantly higher than that in pDC316-tk/lipoplex-transducted ones (semiquantitative analysis, 3.11{+-}0.14 versus 1.60{+-}0.05, P<.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rates were 81.70{+-}0.40% in Ad5-tk and 22.06{+-}0.32% in liposome (P<.01). Conclusions: Both adenovirus and liposome could transfer reporter gene into cardiac myocytes successfully, and the expressed exogenous protein could form functional enzymes efficiently. However, the adenovirus vector acted more efficiently than did liposome, with a higher uptake rate of the reporter probe. Thus, adenovirus is competent for gene transfer in cardiac reporter gene imaging.

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

Science.gov (United States)

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

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De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

2014-01-01

ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins

Czech Academy of Sciences Publication Activity Database

Bachtarzi, H.; Stevenson, M.; Šubr, Vladimír; Ulbrich, Karel; Seymour, L. W.; Fisher, K. D.

2011-01-01

Roč. 150, č. 2 (2011), s. 196-203 ISSN 0168-3659 R&D Projects: GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z40500505 Keywords : E-selectin * pHPMA * adenovirus Subject RIV: EI - Biotechnology ; Bionics Impact factor: 5.732, year: 2011

Oncolytic adenovirus targeting cyclin E overexpression repressed tumor growth in syngeneic immunocompetent mice

International Nuclear Information System (INIS)

Cheng, Pei-Hsin; Rao, Xiao-Mei; Wechman, Stephen L.; Li, Xiao-Feng; McMasters, Kelly M.; Zhou, Heshan Sam

2015-01-01

Clinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads. Ad-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies. Murine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment. Ad-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor. The online version of this article (doi:10.1186/s12885-015-1731-x) contains supplementary material, which is available to authorized users

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

Science.gov (United States)

Lokhandwala, Shehnaz; Waghela, Suryakant D; Bray, Jocelyn; Martin, Cameron L; Sangewar, Neha; Charendoff, Chloe; Shetti, Rashmi; Ashley, Clay; Chen, Chang-Hsin; Berghman, Luc R; Mwangi, Duncan; Dominowski, Paul J; Foss, Dennis L; Rai, Sharath; Vora, Shaunak; Gabbert, Lindsay; Burrage, Thomas G; Brake, David; Neilan, John; Mwangi, Waithaka

2016-11-01

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ + ) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

Science.gov (United States)

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

Science.gov (United States)

Wee, Sunmee; Hicks, Martin J; De, Bishnu P; Rosenberg, Jonathan B; Moreno, Amira Y; Kaminsky, Stephen M; Janda, Kim D; Crystal, Ronald G; Koob, George F

2012-01-01

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>105) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited ‘extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction. PMID:21918504

Profiling the Targets of Protective CD8+ T Cell Responses to Infection

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Joseph T. Bruder

2017-12-01

Full Text Available T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

Science.gov (United States)

Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

2015-08-01

We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

Science.gov (United States)

Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

2003-10-01

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Closing the manufacturing process of dendritic cell vaccines transduced with adenovirus vectors.

Science.gov (United States)

Gulen, Dumrul; Abe, Fuminori; Maas, Sarah; Reed, Elizabeth; Cowan, Kenneth; Pirruccello, Samuel; Wisecarver, James; Warkentin, Phyllis; Northam, Matt; Turken, Orhan; Coskun, Ugur; Senesac, Joe; Talmadge, James E

2008-12-20

Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20

Induction of CD8(+) T cell responses and protective efficacy following microneedle-mediated delivery of a live adenovirus-vectored malaria vaccine.

Science.gov (United States)

Pearson, Frances E; O'Mahony, Conor; Moore, Anne C; Hill, Adrian V S

2015-06-22

There is an urgent need for improvements in vaccine delivery technologies. This is particularly pertinent for vaccination programmes within regions of limited resources, such as those required for adequate provision for disposal of used needles. Microneedles are micron-sized structures that penetrate the stratum corneum of the skin, creating temporary conduits for the needle-free delivery of drugs or vaccines. Here, we aimed to investigate immunity induced by the recombinant simian adenovirus-vectored vaccine ChAd63.ME-TRAP; currently undergoing clinical assessment as a candidate malaria vaccine, when delivered percutaneously by silicon microneedle arrays. In mice, we demonstrate that microneedle-mediated delivery of ChAd63.ME-TRAP induced similar numbers of transgene-specific CD8(+) T cells compared to intradermal (ID) administration with needle-and-syringe, following a single immunisation and after a ChAd63/MVA heterologous prime-boost schedule. When mice immunised with ChAd63/MVA were challenged with live Plasmodium berghei sporozoites, microneedle-mediated ChAd63.ME-TRAP priming demonstrated equivalent protective efficacy as did ID immunisation. Furthermore, responses following ChAd63/MVA immunisation correlated with a specific design parameter of the array used ('total array volume'). The level of transgene expression at the immunisation site and skin-draining lymph node (dLN) was also linked to total array volume. These findings have implications for defining silicon microneedle array design for use with live, vectored vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.

Science.gov (United States)

Saito, Shinta; Ura, Kiyoe; Kodama, Miho; Adachi, Noritaka

2015-06-30

Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

NARCIS (Netherlands)

Roberts, Diane M.; Nanda, Anjali; Havenga, Menzo J. E.; Abbink, Peter; Lynch, Diana M.; Ewald, Bonnie A.; Liu, Jinyan; Thorner, Anna R.; Swanson, Patricia E.; Gorgone, Darci A.; Lifton, Michelle A.; Lemckert, Angelique A. C.; Holterman, Lennart; Chen, Bing; Dilraj, Athmanundh; Carville, Angela; Mansfield, Keith G.; Goudsmit, Jaap; Barouch, Dan H.

2006-01-01

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of

Analysis of the role of homology arms in gene-targeting vectors in human cells.

Directory of Open Access Journals (Sweden)

Ayako Ishii

Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

Transformation and oncogenicity by Adenoviruses

NARCIS (Netherlands)

Bernards, R.A.; Eb, A.J. van der

1984-01-01

Adenoviruses have attracted considerable attention since it was discovered by TRENTIN et all. and HUEBNER et al. that certain species (formerly called serotypes) are oncogenic when injected into newborn hamsters. Since then, adenoviruses have been used extensively as a model for studies on tumor

Gene therapy of cancer and development of therapeutic target gene

Energy Technology Data Exchange (ETDEWEB)

Kim, Chang Min; Kwon, Hee Chung

1998-04-01

We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

Gene therapy of cancer and development of therapeutic target gene

International Nuclear Information System (INIS)

Kim, Chang Min; Kwon, Hee Chung

1998-04-01

We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene

Cryo-EM structures of two bovine adenovirus type 3 intermediates

International Nuclear Information System (INIS)

Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin; Xiong, Wei; Sun, Wei; Yang, Chongwen; Zhang, Kai; Wang, Ying; Liu, Hongrong; Huang, Xiaojun; Ji, Gang; Sun, Fei; Zheng, Congyi; Zhu, Ping

2014-01-01

Adenoviruses (Ads) infect hosts from all vertebrate species and have been investigated as vaccine vectors. We report here near-atomic structures of two bovine Ad type 3 (BAd3) intermediates obtained by cryo-electron microscopy. A comparison between the two intermediate structures reveals that the differences are localized in the fivefold vertex region, while their facet structures are identical. The overall facet structure of BAd3 exhibits a similar structure to human Ads; however, BAd3 protein IX has a unique conformation. Mass spectrometry and cryo-electron tomography analyses indicate that one intermediate structure represents the stage during DNA encapsidation, whilst the other intermediate structure represents a later stage. These results also suggest that cleavage of precursor protein VI occurs during, rather than after, the DNA encapsidation process. Overall, our results provide insights into the mechanism of Ad assembly, and allow the first structural comparison between human and nonhuman Ads at backbone level. - Highlights: • First structure of bovine adenovirus type 3. • Some channels are located at the vertex of intermediate during DNA encapsidation. • Protein IX exhibits a unique conformation of trimeric coiled–coiled structure. • Cleavage of precursor protein VI occurs during the DNA encapsidation process

Cryo-EM structures of two bovine adenovirus type 3 intermediates

Energy Technology Data Exchange (ETDEWEB)

Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xiong, Wei [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Sun, Wei; Yang, Chongwen; Zhang, Kai; Wang, Ying [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Hongrong [College of Physics and Information Science, Hunan Normal University, Changsha, Hunan 410081 (China); Huang, Xiaojun; Ji, Gang; Sun, Fei [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Zheng, Congyi, E-mail: cctcc202@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Zhu, Ping, E-mail: zhup@ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

2014-02-15

Adenoviruses (Ads) infect hosts from all vertebrate species and have been investigated as vaccine vectors. We report here near-atomic structures of two bovine Ad type 3 (BAd3) intermediates obtained by cryo-electron microscopy. A comparison between the two intermediate structures reveals that the differences are localized in the fivefold vertex region, while their facet structures are identical. The overall facet structure of BAd3 exhibits a similar structure to human Ads; however, BAd3 protein IX has a unique conformation. Mass spectrometry and cryo-electron tomography analyses indicate that one intermediate structure represents the stage during DNA encapsidation, whilst the other intermediate structure represents a later stage. These results also suggest that cleavage of precursor protein VI occurs during, rather than after, the DNA encapsidation process. Overall, our results provide insights into the mechanism of Ad assembly, and allow the first structural comparison between human and nonhuman Ads at backbone level. - Highlights: • First structure of bovine adenovirus type 3. • Some channels are located at the vertex of intermediate during DNA encapsidation. • Protein IX exhibits a unique conformation of trimeric coiled–coiled structure. • Cleavage of precursor protein VI occurs during the DNA encapsidation process.

The evolution of heart gene delivery vectors

Science.gov (United States)

Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

2012-01-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

Hexon and fiber of adenovirus type 14 and 55 are major targets of neutralizing antibody but only fiber-specific antibody contributes to cross-neutralizing activity.

Science.gov (United States)

Feng, Ying; Sun, Xikui; Ye, Xianmiao; Feng, Yupeng; Wang, Jinlin; Zheng, Xuehua; Liu, Xinglong; Yi, Changhua; Hao, Mingli; Wang, Qian; Li, Feng; Xu, Wei; Li, Liang; Li, Chufang; Zhou, Rong; Chen, Ling; Feng, Liqiang

2018-05-01

Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic modification of hematopoietic cells using retroviral and lentiviral vectors: safety considerations for vector design and delivery into target cells.

Science.gov (United States)

Dropulic, Boro

2005-07-01

The recent development of leukemia in three patients following retroviral vector gene transfer in hematopoietic stem cells, resulting in the death of one patient, has raised safety concerns for the use of integrating gene transfer vectors for human gene therapy. This review discusses these serious adverse events from the perspective of whether restrictions on vector design and vector-modified target cells are warranted at this time. A case is made against presently establishing specific restrictions for vector design and transduced cells; rather, their safety should be ascertained by empiric evaluation in appropriate preclinical models on a case-by-case basis. Such preclinical data, coupled with proper informed patient consent and a risk-benefit ratio analysis, provide the best available prospective evaluation of gene transfer vectors prior to their translation into the clinic.

21 CFR 866.3020 - Adenovirus serological reagents.

Science.gov (United States)

2010-04-01

... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020 Adenovirus... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease...

A viral vectored prime-boost immunization regime targeting the malaria Pfs25 antigen induces transmission-blocking activity.

Directory of Open Access Journals (Sweden)

Anna L Goodman

Full Text Available The ookinete surface protein Pfs25 is a macrogamete-to-ookinete/ookinete stage antigen of Plasmodium falciparum, capable of exerting high-level anti-malarial transmission-blocking activity following immunization with recombinant protein-in-adjuvant formulations. Here, this antigen was expressed in recombinant chimpanzee adenovirus 63 (ChAd63, human adenovirus serotype 5 (AdHu5 and modified vaccinia virus Ankara (MVA viral vectored vaccines. Two immunizations were administered to mice in a heterologous prime-boost regime. Immunization of mice with AdHu5 Pfs25 at week 0 and MVA Pfs25 at week 10 (Ad-MVA Pfs25 resulted in high anti-Pfs25 IgG titers, consisting of predominantly isotypes IgG1 and IgG2a. A single priming immunization with ChAd63 Pfs25 was as effective as AdHu5 Pfs25 with respect to ELISA titers at 8 weeks post-immunization. Sera from Ad-MVA Pfs25 immunized mice inhibited the transmission of P. falciparum to the mosquito both ex vivo and in vivo. In a standard membrane-feeding assay using NF54 strain P. falciparum, oocyst intensity in Anopheles stephensi mosquitoes was significantly reduced in an IgG concentration-dependent manner when compared to control feeds (96% reduction of intensity, 78% reduction in prevalence at a 1 in 5 dilution of sera. In addition, an in vivo transmission-blocking effect was also demonstrated by direct feeding of immunized mice infected with Pfs25DR3, a chimeric P. berghei line expressing Pfs25 in place of endogenous Pbs25. In this assay the density of Pfs25DR3 oocysts was significantly reduced when mosquitoes were fed on vaccinated as compared to control mice (67% reduction of intensity, 28% reduction in prevalence and specific IgG titer correlated with efficacy. These data confirm the utility of the adenovirus-MVA vaccine platform for the induction of antibodies with transmission-blocking activity, and support the continued development of this alternative approach to transmission-blocking malaria subunit

Adenovirus sequences required for replication in vivo.

OpenAIRE

Wang, K; Pearson, G D

1985-01-01

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

Adenovirus-Mediated Delivery of Decoy Hyper Binding Sites Targeting Oncogenic HMGA1 Reduces Pancreatic and Liver Cancer Cell Viability.

Science.gov (United States)

Hassan, Faizule; Ni, Shuisong; Arnett, Tyler C; McKell, Melanie C; Kennedy, Michael A

2018-03-30

High mobility group AT-hook 1 (HMGA1) protein is an oncogenic architectural transcription factor that plays an essential role in early development, but it is also implicated in many human cancers. Elevated levels of HMGA1 in cancer cells cause misregulation of gene expression and are associated with increased cancer cell proliferation and increased chemotherapy resistance. We have devised a strategy of using engineered viruses to deliver decoy hyper binding sites for HMGA1 to the nucleus of cancer cells with the goal of sequestering excess HMGA1 at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA1 at the decoy binding sites is intended to reduce HMGA1 binding at the naturally occurring genomic HMGA1 binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. As proof of principle, we engineered the replication defective adenovirus serotype 5 genome to contain hyper binding sites for HMGA1 composed of six copies of an individual HMGA1 binding site, referred to as HMGA-6. A 70%-80% reduction in cell viability and increased sensitivity to gemcitabine was observed in five different pancreatic and liver cancer cell lines 72 hr after infection with replication defective engineered adenovirus serotype 5 virus containing the HMGA-6 decoy hyper binding sites. The decoy hyper binding site strategy should be general for targeting overexpression of any double-stranded DNA-binding oncogenic transcription factor responsible for cancer cell proliferation.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

Science.gov (United States)

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

International Nuclear Information System (INIS)

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

2012-01-01

Highlights: ► Adeno-associated virus (AAV) is capable of targeted integration in human cells. ► Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. ► A targeted integration system of IDRV DNA using the AAV integration mechanism. ► Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats : preferential survival of transduced astroglial cells in nude rats

NARCIS (Netherlands)

Hermens, W.T.J.M.C.; Verhaagen, J

1997-01-01

In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

Directory of Open Access Journals (Sweden)

Kenta Kobayashi

2017-08-01

Full Text Available Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1 with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G and vesicular stomatitis virus glycoprotein (VSV-G enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E, which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease.

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

Science.gov (United States)

Tollefson, A E; Toth, K; Doronin, K; Kuppuswamy, M; Doronina, O A; Lichtenstein, D L; Hermiston, T W; Smith, C A; Wold, W S

2001-10-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

Directory of Open Access Journals (Sweden)

Chitose Kurihara

2016-12-01

Full Text Available The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL. This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre. Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF into induced-hepatocyte-like cells (iHep following several rounds of infection, demonstrating the efficacy of this new system.

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

Science.gov (United States)

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery.

Science.gov (United States)

Kaulfuß, Meike; Wensing, Ina; Windmann, Sonja; Hrycak, Camilla Patrizia; Bayer, Wibke

2017-02-06

In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines that were designed to induce either strong Friend virus GagL 85-93 -specific CD8 + T cell or antibody responses, respectively. To optimize the immunization outcome we evaluated vaccination strategies using combinations of these vaccines. While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine effectivity. We demonstrate that the co-immunization with GagL 85-93 /leader-gag encoding vectors together with envelope-encoding vectors abrogates the induction of GagL 85-93 -specific CD8 + T cells, and in successive immunization protocols the immunization with the GagL 85-93 /leader-gag encoding vector had to precede the immunization with an envelope encoding vector for the efficient induction of GagL 85-93 -specific CD8 + T cells. Importantly, the antibody response to envelope was in fact enhanced when the mice were adenovirus-experienced from a prior immunization, highlighting the expedience of this approach. To circumvent the immunosuppressive effect of envelope on immune responses to simultaneously or subsequently administered immunogens, we developed a two immunizations-based vaccination protocol that induces strong immune responses and confers robust protection of highly Friend virus-susceptible mice from a lethal Friend virus challenge.

The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

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Shuhua Li

Full Text Available We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5 particles into matrix metalloproteinase (MMP-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N. ACPPs are activatable cell penetrating peptides (CPPs with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

T Helper 17 Promotes Induction of Antigen-Specific Gut-Mucosal Cytotoxic T Lymphocytes following Adenovirus Vector Vaccination

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Masahisa Hemmi

2017-11-01

Full Text Available Few current vaccines can establish antigen (Ag-specific immune responses in both mucosal and systemic compartments. Therefore, development of vaccines providing defense against diverse infectious agents in both compartments is of high priority in global health. Intramuscular vaccination of an adenovirus vector (Adv has been shown to induce Ag-specific cytotoxic T lymphocytes (CTLs in both systemic and gut-mucosal compartments. We previously found that type I interferon (IFN signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that T helper 17 (Th17-polarizing cytokine expression was down-regulated in the inguinal lymph nodes (iLNs of Ifnar2−/− mice, resulting in the reduction of Ag-specific Th17 cells in the iLNs and gut mucosa of the mice. We also found that prior transfer of Th17 cells reversed the decrease in the number of Ag-specific gut-mucosal CTLs in Ifnar2−/− mice following Adv vaccination. Additionally, prior transfer of Th17 cells into wild-type mice enhanced the induction of Ag-specific CTLs in the gut mucosa, but not in systemic compartments, suggesting a gut mucosa-specific mechanism where Th17 cells regulate the magnitude of vaccine-elicited Ag-specific CTL responses. These data suggest that Th17 cells translate systemic type I IFN signaling into a gut-mucosal CTL response following vaccination, which could promote the development of promising Adv vaccines capable of establishing both systemic and gut-mucosal protective immunity.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

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Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

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Lopez-Gordo, Estrella; Podgorski, Iva I; Downes, Nicholas; Alemany, Ramon

2014-04-01

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.

Protective Immunity against Tularemia Provided by an Adenovirus-Vectored Vaccine Expressing Tul4 of Francisella tularensis

OpenAIRE

Kaur, Ravinder; Chen, Shan; Arévalo, Maria T.; Xu, Qingfu; Chen, Yanping; Zeng, Mingtao

2012-01-01

Francisella tularensis, a category A bioterrorism agent, is a highly infectious organism that is passed on via skin contact and inhalation routes. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication-incompetent adenovirus, Ad/opt-Tul4, carrying a codon-optimized gene for expression of a membrane prote...

Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

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Kamola Saydaminova

Full Text Available Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35 vectors for zinc-finger nuclease (ZFN– or transcription activator-like effector nuclease (TALEN–mediated genome editing in human CD34+ hematopoietic stem cells (HSCs from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells. The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2 within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

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Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam

2018-06-06

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative inactivation of enteric adenoviruses, poliovirus and coliphages by ultraviolet irradiation

International Nuclear Information System (INIS)

Meng, Q.S.; Gerba, C.P.

1996-01-01

The inactivation of enteric adenoviruses 40 and 41 by ultraviolet (UV) radiation was investigated and compared with poliovirus type 1 (strain LSc-2ab) and coliphages MS-2 and PRD-1. Purified stocks of the viruses were exposed to collimated ultraviolet radiation in a stirred reactor for a total dose of up to 140 mW s/cm 2 . The doses of UV to achieve a 90% inactivation of adenovirus 40, adenovirus 41, coliphages MS-2 and PRD-1 and poliovirus type 1 were 30, 23.6, 14, 8.7 and 4.1 mW s/cm 2 , respectively. Adenovirus 40 was significantly more resistant than coliphage MS-2 to UV irradiation (P < 0.01). Adenovirus 41 appeared slightly more sensitive than adenovirus 40, but the difference was not significant (P>0.05). The resistance of PRD-1 was less than MS-2 (P < 0.01), but greater than poliovirus type 1 (P < 0.01). Adenoviruses 40 and 41 were more resistant than Bacillus subtilis spores, often suggested as an indicator of UV light performance. The double-stranded DNA adenoviruses appear to be the most resistant of all potentially water-borne enteric viruses to UV light disinfection. (author)

Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors

NARCIS (Netherlands)

Verhaagh, S.; Jong, E. de; Goudsmit, J.; Lecollinet, S.; Gillissen, G.; Vries, M. de; Leuven, K. van; Que, I.; Ouwehand, K.; Mintardjo, R.; Weverling, G.J.; Radošević, K.; Richardson, J.; Eloit, M.; Lowik, C.; Quax, P.; Havenga, M.

2006-01-01

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here,

Adenovirus-based vaccine against Listeria monocytogenes

DEFF Research Database (Denmark)

Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech

2013-01-01

The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC...... linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further...

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.

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Khanam, Saima; Rajendra, Pilankatta; Khanna, Navin; Swaminathan, Sathyamangalam

2007-02-15

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

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Swaminathan Sathyamangalam

2007-02-01

Full Text Available Abstract Background Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. Results This work stems from the emergence of (i the DEN virus envelope (E domain III (EDIII as the most important region of the molecule from a vaccine perspective and (ii the adenovirus (Ad as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Conclusion Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has

Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

International Nuclear Information System (INIS)

Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E.; Leeuwen, M. van; Boheemen, S. van; Jong, A.A.W. de; Osterhaus, A.D.M.E.; Smits, S.L.; Kuiken, T.

2013-01-01

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. - Highlights: ► Lesions typical for adenovirus infection detected in cloacal bursa of dead gulls. ► Confirmation of adenovirus infection by electron microscopy and deep sequencing. ► Sequence analysis indicates that it is a novel adenovirus in the genus Aviadenovirus. ► The novel (Gull) adenovirus was detected in multiple organs of two species of gulls

Immunogenicity and efficacy in mice of an adenovirus-based bicistronic rotavirus vaccine expressing NSP4 and VP7.

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Xie, Li; Yan, Min; Wang, Xiaonan; Ye, Jing; Mi, Kai; Yan, Shanshan; Niu, Xianglian; Li, Hongjun; Sun, Maosheng

2015-12-02

NSP4 and VP7 are important functional proteins of rotavirus. Proper combination of viral gene expression is favorable to improving the protection effect of subunit vaccine. In the present study, We evaluated the immunogenicity and efficacy of the bicistronic recombinant adenovirus (rAd-NSP4-VP7) and two single-gene expressing adenoviruses (rAd-NSP4, rAd-VP7). The three adenovirus vaccines were used to immunize mice by intramuscular or intranasal administration. The data showed significant increases in serum antibodies, T lymphocyte subpopulations proliferation, and cytokine secretions of splenocyte in all immunized groups. However, the serum IgA and neutralizing antibody levels of the rAd-NSP4-VP7 or rAd-VP7 groups were significantly higher than those of the rAd-NSP4, while the splenocyte numbers of IFN-γ secretion in the rAd-NSP4-VP7 or rAd-NSP4 groups was greater than that of the rAd-VP7. Furthermore, the efficacy evaluation in a suckling mice model indicated that only rAd-NSP4-VP7 conferred significant protection against rotavirus shedding challenge. These results suggest that the co-expression of NSP4 and VP7 in an adenovirus vector induce both humoral and cell-mediated immune responses efficiently, and provide potential efficacy for protection against rotavirus disease. It is possible to represent an efficacious subunits vaccine strategy for control of rotavirus infection and transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

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Motoharu Ono

Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

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Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

2015-01-01

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

Viral vectors for cystic fibrosis gene therapy: What does the future hold?

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Uta Griesenbach

2010-12-01

Full Text Available Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

Oncolytic adenovirus Ad657 for systemic virotherapy against prostate cancer

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Nguyen TV

2018-05-01

Full Text Available Tien V Nguyen,1,* Catherine M Crosby,2,* Gregory J Heller,3 Zachary I Mendel,3 Mary E Barry,1 Michael A Barry1,4,5 1Department of Internal Medicine, Division of Infectious Diseases, 2Virology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, 3Postbaccalaureate Research Education Program, Mayo Clinic Graduate School of Biomedical Sciences, 4Department of Immunology, 5Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA *These authors contributed equally to this work Background: Human species C adenovirus serotype 5 (Ad5 is the archetype oncolytic adenovirus and has been used in the vast majority of preclinical and clinical tests. While Ad5 can be robust, species C Ad6 has lower seroprevalence, side effects, and appears to be more potent as a systemic therapy against a number of tumors than Ad5. Historically, there have only been four species C human adenoviruses: serotypes 1, 2, 5, and 6. More recently a new species C adenovirus, Ad57, was identified. Ad57 is most similar to Ad6 with virtually all variation in their capsid proteins occurring in the hypervariable regions (HVRs of their hexon proteins. Most adenovirus neutralizing antibodies target the HVRs on adenoviruses. This led us to replace the hexon HVRs in Ad6 with those from Ad57 to create a new virus called Ad657 and explore this novel species C platform’s utility as an oncolytic virus. Methods: The HVR region from Ad57 was synthesized and used to replace the Ad6 HVR region by homologous recombination in bacteria generating a new viral platform that we call Ad657. Replication-competent Ad5, Ad6, and Ad657 were compared in vitro and in vivo for liver damage and oncolytic efficacy against prostate cancers after single intravenous treatment in mice. Results: Ad5, Ad6, and Ad657 had similar in vitro oncolytic activity against human prostate cancer cells. Ad5 provoked the highest level of liver toxicity after intravenous injection and Ad657

The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

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Kathleen Kong

2014-05-01

Full Text Available Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K. While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1, a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.

In Vitro Transduction and Target-Mutagenesis Efficiency of HIV-1 pol Gene Targeting ZFN and CRISPR/Cas9 Delivered by Various Plasmids and/or Vectors: Toward an HIV Cure.

Science.gov (United States)

Okee, Moses; Bayiyana, Alice; Musubika, Carol; Joloba, Moses L; Ashaba-Katabazi, Fred; Bagaya, Bernard; Wayengera, Misaki

2018-01-01

Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate. Cell lines were grown in either Dulbecco's modified Eagle's medium or Roswell Park Memorial Institute with the antibiotics kanamycin, zeocin, and efavirenz. Efficiency was assayed by GFP/luciferase activity and/or validated by yeast MEL1 reporter assay, CEL1 restriction fragment assay, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Ad5-RGD vectors had better transduction efficiency than murCTSD and pGS-U-gRNA/pCMV-Cas-D01A plasmids. CRISPR/Cas9 exhibited better target-mutagenesis efficiency relative to ZFN (delivered by either plasmid or Ad5 vector) based on gel electrophoresis of pol gene amplicons within ACH-2 and J-Lat cells. Ad-5-RGD vectors enhanced target mutagenesis of ZFN, relative to murCTSD_pZFN plasmids, to levels of CRISPR/Cas9 plasmids. Similar reduction of luciferase activity among TZM-bl treated with Ad5-ZFN vectors relative to CRISPR/Cas-9 and murCTSD_pZFN plasmids was observed on challenge with HIV-1. qRT-PCR of HIV-1 pol gene transcripts affirmed that Ad5 (RGD) vectors enhanced target mutagenesis of ZFN. Whereas CRISPR/Cas-9 may possess inherent superior target-mutagenesis efficiency; the efficiency of ZFN (off-target toxicity withstanding) can be enhanced by altering delivery vehicle from plasmid to Ad5 (RGD) vectors.

A dynamical systems model for combinatorial cancer therapy enhances oncolytic adenovirus efficacy by MEK-inhibition.

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Bagheri, Neda; Shiina, Marisa; Lauffenburger, Douglas A; Korn, W Michael

2011-02-01

Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced

Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

International Nuclear Information System (INIS)

Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine

2006-01-01

To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours

Link of the unique oncogenic properties of adenovirus type 9 E4-ORF1 to a select interaction with the candidate tumor suppressor protein ZO-2

OpenAIRE

Glaunsinger, Britt A.; Weiss, Robert S.; Lee, Siu Sylvia; Javier, Ronald

2001-01-01

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region-encoded ORF1 (E4-ORF1) protein. We report here that the PDZ domain-containing protein ZO-2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4-ORF1 but not for non-tumorigenic wild-type E4-ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C-terminal...

Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

International Nuclear Information System (INIS)

Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

2004-01-01

For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

Adenovirus (For Parents)

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... by sharing contaminated objects (such as towels or toys), or by touch. Once a child is exposed to adenovirus, symptoms usually develop from ... washing, keep shared surfaces (such as countertops and toys) clean, and remove kids ... a week your child has breathing problems your child is under 3 ...

ENTERIC ADENOVIRUS INFECTION IN INFANTS AND YOUNG CHILDREN WITH ACUTE GASTROENTERITIS IN TEHRAN

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F. Jam-Afzon S. Modarres

2006-09-01

Full Text Available Adenoviruses are one of the most important etiological agents of serious gastroenteritis among infants and young children. Fecal specimens from patients with an acute gastroenteritis were evaluated for the presence of adenovirus (Ad40, 41 from April 2002 to February 2004. During the study, 1052 samples were collected from children under the age of 5 years in six educational and therapeutic pediatric centers. The specimens were tested for adenovirus (Ad40, 41 by EIA technique in the Virology Department of Pasteur Institute of Iran. Adenoviruses (Ad40, 41 were detected from 27(2.6% samples, but were not detected in 150 samples of healthy control group. In this study the highest rate of adenovirus was found in children aged 6 to 12 months (40.7%, but the male to female ratio inpatients was approximately equal. Adenovirus (Ad40, 41 infections peaked in the winter as 48.1% was detected from December to March. There were a statistically significant difference between age and infection (P < 0.001, also between season with adenovirus (Ad40, 41 infection (P = 0.005. Breast-feeding had a protective action against adenovirus (Ad40, 41 infection. This study revealed that enteric adenovirus (Ad40, 41 is an etiological agent of acute gastroenteritis among children in Tehran.

The experimental study of reporter probe 131I-FIAU in neonatal cardiac myocytes after transfer of herpes simplex virus type 1 thymidine kinase reporter gene by different vectors

International Nuclear Information System (INIS)

Yin Xiaohua; Lan Xiaoli; Wang Ruihua; Liu Ying; Zhang Yongxue

2009-01-01

accumulation of 131 I-F1AU for Ad5-tk infected cells compared with pDC316-tk/lipoplex transfected ones occurred for all the time points (t=12.978-38.253. all P<0.01). When 24h after transferred. the cardiac myocytes showed significant expression of HSVI-tk mRNA. while the control groups showed negative. Moreover, the exogenous gene expression in the adenovirus vector-infected cardiac. myocytes was significantly higher than in the pDC316-tk/lipoplex-transducted ones (3.11 ± 0.14 vs1.60 ± 0.05, t=17.663, P<0.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rate was (81.70 ± 0.40)% and (22.06 ± 0.32)% respectively, and there was also significant difference between them (t=23.237, P<0.01). Conclusions: Both vectors could transfer the reporter gene into the cardiac myocytes successfully, and the expressed exogenous protein could form a functional enzyme as to uptake the reporter probe much efficiently. But the transferred cardiac myocytes mediated by the adenovirus vector acted even more efficiently than the ones mediated by liposome-coated plasmid with higher uptake rate. Thus the adenovirus was competent for the gene transfer in cardiac reporter gene imaging. (authors)

Adenovirus type 35-vectored tuberculosis vaccine has an acceptable safety and tolerability profile in healthy, BCG-vaccinated, QuantiFERON(®)-TB Gold (+) Kenyan adults without evidence of tuberculosis.

Science.gov (United States)

Walsh, Douglas S; Owira, Victorine; Polhemus, Mark; Otieno, Lucas; Andagalu, Ben; Ogutu, Bernhards; Waitumbi, John; Hawkridge, Anthony; Shepherd, Barbara; Pau, Maria Grazia; Sadoff, Jerald; Douoguih, Macaya; McClain, J Bruce

2016-05-05

In a Phase 1 trial, we evaluated the safety of AERAS-402, an adenovirus 35-vectored TB vaccine candidate expressing 3 Mycobacterium tuberculosis (Mtb) immunodominant antigens, in subjects with and without latent Mtb infection. HIV-negative, BCG-vaccinated Kenyan adults without evidence of tuberculosis, 10 QuantiFERON(®)-TB Gold In-Tube test (QFT-G)(-) and 10 QFT-G(+), were randomized 4:1 to receive AERAS-402 or placebo as two doses, on Days 0 and 56, with follow up to Day 182. There were no deaths, serious adverse events or withdrawals. For 1 AERAS-402 QFT-G(-) and 1 AERAS-402 QFT-G(+) subject, there were 3 self-limiting severe AEs of injection site pain: 1 after the first vaccination and 1 after each vaccination, respectively. Two additional severe AEs considered vaccine-related were reported after the first vaccination in AERAS-402 QFT-G(+) subjects: elevated blood creatine phosphokinase and neutropenia, the latter slowly improving but remaining abnormal until study end. AERAS-402 was not detected in urine or throat cultures for any subject. In intracellular cytokine staining studies, curtailed by technical issues, we saw modest CD4+ and CD8+ T cell responses to Mtb Ag85A/b peptide pools among both QFT-G(-) and (+) subjects, with trends in the CD4+ T cells suggestive of boosting after the second vaccine dose, slightly more so in QFT-G(+) subjects. CD4+ and CD8+ responses to Mtb antigen TB10.4 were minimal. Increases in Adenovirus 35 neutralizing antibodies from screening to end of study, seen in 50% of AERAS-402 recipients, were mostly minimal. This small study confirms acceptable safety and tolerability profiles for AERAS-402, in line with other Phase 1 studies of AERAS-402, now to include QFT-G(+) subjects. Published by Elsevier Ltd.

Redirecting adenovirus tropism by genetic, chemical, and mechanical modification of the adenovirus surface for cancer gene therapy.

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Yoon, A-Rum; Hong, Jinwoo; Kim, Sung Wan; Yun, Chae-Ok

2016-06-01

Despite remarkable advancements, clinical evaluations of adenovirus (Ad)-mediated cancer gene therapies have highlighted the need for improved delivery and targeting. Genetic modification of Ad capsid proteins has been extensively attempted. Although genetic modification enhances the therapeutic potential of Ad, it is difficult to successfully incorporate extraneous moieties into the capsid and the engineering process is laborious. Recently, chemical modification of the Ad surface with nanomaterials and targeting moieties has been found to enhance Ad internalization into the target by both passive and active mechanisms. Alternatively, external stimulus-mediated targeting can result in selective accumulation of Ad in the tumor and prevent dissemination of Ad into surrounding nontarget tissues. In the present review, we discuss various genetic, chemical, and mechanical engineering strategies for overcoming the challenges that hinder the therapeutic efficacy of Ad-based approaches. Surface modification of Ad by genetic, chemical, or mechanical engineering strategies enables Ad to overcome the shortcomings of conventional Ad and enhances delivery efficiency through distinct and unique mechanisms that unmodified Ad cannot mimic. However, although the therapeutic potential of Ad-mediated gene therapy has been enhanced by various surface modification strategies, each strategy still possesses innate limitations that must be addressed, requiring innovative ideas and designs.

Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against Zika Virus Infection and Testis Damage.

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Xu, Kun; Song, Yufeng; Dai, Lianpan; Zhang, Yongli; Lu, Xuancheng; Xie, Yijia; Zhang, Hangjie; Cheng, Tao; Wang, Qihui; Huang, Qingrui; Bi, Yuhai; Liu, William J; Liu, Wenjun; Li, Xiangdong; Qin, Chuan; Shi, Yi; Yan, Jinghua; Zhou, Dongming; Gao, George F

2018-03-15

The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitoes, as seen for classical arboviruses. We along with others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available; therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low preexisting immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week postvaccination, and the elicited antiserum could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mouse sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control. IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barré syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid preexisting anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M

Presence of adenovirus species C in infiltrating lymphocytes of human sarcoma.

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Karin Kosulin

Full Text Available Human adenoviruses are known to persist in T-lymphocytes of tonsils, adenoids and intestinal tract. The oncogenic potential of different adenovirus types has been widely studied in rodents, in which adenovirus inoculation can induce multiple tumors such as undifferentiated sarcomas, adenocarcinomas and neuroectodermal tumors. However, the oncogenic potential of this virus has never been proven in human subjects. Using a highly sensitive broad-spectrum qRT-PCR, we have screened a set of different human sarcomas including leiomyosarcoma, liposarcoma and gastro intestinal stroma tumors. Primers binding the viral oncogene E1A and the capsid-coding gene Hexon were used to detect the presence of adenovirus DNA in tumor samples. We found that 18% of the tested leiomyosarcomas and 35% of the liposarcomas were positive for the presence of adenovirus DNA, being species C types the most frequently detected adenoviruses. However, only in one sample of the gastro intestinal stroma tumors the virus DNA could be detected. The occurrence of adenovirus in the tumor sections was confirmed by subsequent fluorescence in-situ-hybridization analysis and co-staining with the transcription factor Bcl11b gives evidence for the presence of the virus in infiltrating T-lymphocytes within the tumors. Together these data underline, for the first time, the persistence of adenovirus in T-lymphocytes infiltrated in muscular and fatty tissue tumor samples. If an impaired immune system leads to the viral persistence and reactivation of the virus is involved in additional diseases needs further investigation.

Bioaccumulation of animal adenoviruses in the pink shrimp

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Roger B. Luz

2015-09-01

Full Text Available Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100 Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells.

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Sadhak Sengupta

2011-03-01

Full Text Available Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR. T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD.A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

Kernel method for clustering based on optimal target vector

International Nuclear Information System (INIS)

Angelini, Leonardo; Marinazzo, Daniele; Pellicoro, Mario; Stramaglia, Sebastiano

2006-01-01

We introduce Ising models, suitable for dichotomic clustering, with couplings that are (i) both ferro- and anti-ferromagnetic (ii) depending on the whole data-set and not only on pairs of samples. Couplings are determined exploiting the notion of optimal target vector, here introduced, a link between kernel supervised and unsupervised learning. The effectiveness of the method is shown in the case of the well-known iris data-set and in benchmarks of gene expression levels, where it works better than existing methods for dichotomic clustering

Phylogenetic and pathogenic characterization of novel adenoviruses from long-tailed ducks (Clangula hyemalis)

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Counihan, Katrina; Skerratt, Lee; Franson, J. Christian; Hollmen, Tuula E.

2015-01-01

Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis

Science.gov (United States)

Wang, Danher; Suhrbier, Andreas; Penn-Nicholson, Adam; Woraratanadharm, Jan; Gardner, Joy; Luo, Min; Le, Thuy T.; Anraku, Itaru; Sakalian, Michael; Einfeld, David; Dong, John Y.

2011-01-01

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates. PMID:21320541

A single exercise bout augments adenovirus-specific T-cell mobilization and function.

Science.gov (United States)

Kunz, Hawley E; Spielmann, Guillaume; Agha, Nadia H; O'Connor, Daniel P; Bollard, Catherine M; Simpson, Richard J

2018-04-30

Adoptive transfer of virus-specific T-cells (VSTs) effectively treats viral infections following allogeneic hematopoietic stem cell transplantation (alloHSCT), but logistical difficulties have limited widespread availability of VSTs as a post-transplant therapeutic. A single exercise bout mobilizes VSTs specific for latent herpesviruses (i.e. CMV and EBV) to peripheral blood and augments their ex vivo expansion. We investigated whether exercise exerts similar effects on T-cells specific for a NON-latent virus such as adenovirus, which is a major contributor to infection-related morbidity and mortality after alloHSCT. Thirty minutes of cycling exercise increased circulating adenovirus-specific T-cells 2.0-fold and augmented their ex vivo expansion by ~33% compared to rest without altering antigen and MHC-specific autologous target cell killing capabilities. We conclude that exercise is a simple and economical adjuvant to boost the isolation and manufacture of therapeutic VSTs specific to latent and non-latent viruses from healthy donors. Copyright © 2018. Published by Elsevier Inc.

Radar target classification method with high accuracy and decision speed performance using MUSIC spectrum vectors and PCA projection

Science.gov (United States)

Secmen, Mustafa

2011-10-01

This paper introduces the performance of an electromagnetic target recognition method in resonance scattering region, which includes pseudo spectrum Multiple Signal Classification (MUSIC) algorithm and principal component analysis (PCA) technique. The aim of this method is to classify an "unknown" target as one of the "known" targets in an aspect-independent manner. The suggested method initially collects the late-time portion of noise-free time-scattered signals obtained from different reference aspect angles of known targets. Afterward, these signals are used to obtain MUSIC spectrums in real frequency domain having super-resolution ability and noise resistant feature. In the final step, PCA technique is applied to these spectrums in order to reduce dimensionality and obtain only one feature vector per known target. In the decision stage, noise-free or noisy scattered signal of an unknown (test) target from an unknown aspect angle is initially obtained. Subsequently, MUSIC algorithm is processed for this test signal and resulting test vector is compared with feature vectors of known targets one by one. Finally, the highest correlation gives the type of test target. The method is applied to wire models of airplane targets, and it is shown that it can tolerate considerable noise levels although it has a few different reference aspect angles. Besides, the runtime of the method for a test target is sufficiently low, which makes the method suitable for real-time applications.

Heterologous Two-Dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-Lasting Cellular Immunity to Influenza Virus A in Healthy Adults

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L. Coughlan

2018-03-01

Full Text Available Background: T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP and matrix protein-1 (M1. We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. Methods: We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18–46 years and 24 subjects aged 50 years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18 months. Findings: Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP + M1 vaccination was significantly higher compared to ChAdOx1 NP + M1. In a mixed regression model, T-cell responses over 18 months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP + M1. Interpretation: A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP + M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. Funding Source: Medical Research Council UK, NIHR BMRC Oxford. Keywords: Influenza, T-cell responses, Influenza vaccines, Viral vectors, Adults, Older adults

Broad-aperture polarized proton target with arbitrary orientation of polarization vector

International Nuclear Information System (INIS)

Belyaev, A.A.; Get'man, V.A.; Derkach, A.Ya.; Karnaukhov, I.M.; Lukhanin, A.A.; Razumnyj, A.A.; Sorokin, P.V.; Sporov, E.A.; Telegin, Yu.N.; Trotsenko, V.I.

1985-01-01

Polarized proton target with the Helmholtz broad-aperture superconducting magnetic system is described. Axial aperture α=95 deg, inter-coil access angle β=23 deg. The structure of the target allows various versions of the installation what make sure an arbitrary orientation of polarization vector. The 0.1 W cold output 3 He evaporation cryostat was used to obtain the work temperature 0.5 K allowing quick transformation to a 3 He- 4 He dilution refrigerator. Results of the study are given on the dynamical proton polarization in 1,2-propylenglycol with various stable Cr 5 complexes

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

Science.gov (United States)

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Chikungunya Virus Vaccines: Viral Vector-Based Approaches.

Science.gov (United States)

Ramsauer, Katrin; Tangy, Frédéric

2016-12-15

In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

HIV-derived vectors for gene therapy targeting dendritic cells.

Science.gov (United States)

Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

2013-01-01

Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

Science.gov (United States)

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

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Poornima L N Kotha

2015-03-01

Full Text Available Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR, a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

Identification of a novel aviadenovirus, designated pigeon adenovirus 2 in domestic pigeons (Columba livia).

Science.gov (United States)

Teske, L; Rubbenstroth, D; Meixner, M; Liere, K; Bartels, H; Rautenschlein, S

2017-01-02

The young pigeon disease syndrome (YPDS) affects mainly young pigeons of less than one year of age and leads to crop stasis, vomitus, diarrhea, anorexia and occasionally death. This disease is internationally a major health problem because of its seasonal appearance during competitions such as homing pigeon races or exhibitions of ornamental birds. While the etiology of YPDS is still unclear, adenoviruses are frequently discussed as potential causative agents. Electron microscopy of feces from a YPDS outbreak revealed massive shedding of adenovirus-like particles. Whole genome sequencing of this sample identified a novel adenovirus tentatively named pigeon adenovirus 2 (PiAdV-2). Phylogenetic and comparative genome analysis suggest PiAdV-2 to belong to a new species within the genus Aviadenovirus, for which we propose the name Pigeon aviadenovirus B. The PiAdV-2 genome shares 54.9% nucleotide sequence identity with pigeon adenovirus 1 (PiAdV-1). In a screening of further YPDS-affected flocks two variants of PiAdV-2 (variant A and B) were detected which shared 97.6% nucleotide identity of partial polymerase sequences, but only 79.7% nucleotide identity of partial hexon sequences. The distribution of both PiAdV-2 variants was further investigated in fecal samples collected between 2008 and 2015 from healthy or YPDS-affected racing pigeons of different lofts. Independent of their health status, approximately 20% of young and 13% of adult pigeon flocks harbored PiAdV-2 variants. Birds were free of PiAdV-1 or other aviadenoviruses as determined by PCRs targeting the aviadenovirus polymerase or the PiAdV-1 fiber gene, respectively. In conclusion, there is no indication of a correlation between YPDS outbreaks and the presence of PiAdV-2 or other aviadenoviruses, arguing against an causative role in this disease complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Vaxvec: The first web-based recombinant vaccine vector database and its data analysis

Science.gov (United States)

Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

2015-01-01

A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development. PMID:26403370

A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

Science.gov (United States)

Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

2012-01-01

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5–based Constructs

Science.gov (United States)

Alonso-Padilla, Julio; Papp, Tibor; Kaján, Győző L; Benkő, Mária; Havenga, Menzo; Lemckert, Angelique; Harrach, Balázs; Baker, Andrew H

2016-01-01

Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes. PMID:26478249

Fluorescence-guided surgery of a highly-metastatic variant of human triple-negative breast cancer targeted with a cancer-specific GFP adenovirus prevents recurrence

Science.gov (United States)

Yano, Shuya; Takehara, Kiyoto; Miwa, Shinji; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Urata, Yasuo; Kagawa, Shunsuke; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2016-01-01

We have previously developed a genetically-engineered GFP-expressing telomerase-dependent adenovirus, OBP-401, which can selectively illuminate cancer cells. In the present report, we demonstrate that targeting a triple-negative high-invasive human breast cancer, orthotopically-growing in nude mice, with OBP-401 enables curative fluorescence-guided surgery (FGS). OBP-401 enabled complete resection and prevented local recurrence and greatly inhibited lymph-node metastasis due to the ability of the virus to selectively label and subsequently kill cancer cells. In contrast, residual breast cancer cells become more aggressive after bright (white)-light surgery (BLS). OBP-401-based FGS also improved the overall survival compared with conventional BLS. Thus, metastasis from a highly-aggressive triple-negative breast cancer can be prevented by FGS in a clinically-relevant mouse model. PMID:27689331

Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

Science.gov (United States)

Kardinal, C; Selmayr, M; Mocikat, R

1996-11-01

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation.

Early RNA of adenovirus type 3 in permissive and abortive infections.

OpenAIRE

Groff, D E; Daniell, E

1981-01-01

Early adenovirus type 3 cytoplasmic polyadenylated RNAs from HeLa and BHK-21 cells were detected and mapped on the viral genome by gel blotting and hybridization techniques. The sizes and locations of the 16 adenovirus type 3 RNAs were identical in the two cell types, although relative molarities of the various RNA species differed. Each of the early adenovirus type 3 RNAs was associated with polysomes in both cell types, suggesting that the abortive infection of hamster cells does not result...

Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

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Groitl Peter

2011-09-01

Full Text Available Abstract Background Type I interferons (IFNs exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV or HIV. Results Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4+ T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4+ T cell responses were enhanced by IFNα subtypes. Conclusions Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4+ T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines.

Progress on adenovirus-vectored universal influenza vaccines

OpenAIRE

Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

2015-01-01

Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8+ T cell responses targeting viral internal proteins nucleoprotein ...

Immunology and evolvement of the adenovirus prime, MVA boost Ebola virus vaccine.

Science.gov (United States)

Zhou, Yan; Sullivan, Nancy J

2015-08-01

The 2014 Ebola virus outbreak caused an order of magnitude more deaths in a single outbreak than all previous known outbreaks combined, affecting both local and international public health, and threatening the security and economic stability of the countries in West Africa directly confronting the outbreak. The severity of the epidemic lead to a global response to assist with patient care, outbreak control, and deployment of vaccines. The latter was possible due to the long history of basic and clinical research aimed at identifying a safe and effective vaccine to protect against Ebola virus infection. This review highlights the immunology, development, and progress of vaccines based on replication-defective adenovirus vectors, culminating in the successful launch of the first Phase III trial of an Ebola virus vaccine. Published by Elsevier Ltd.

Novel Infectivity-Enhanced Oncolytic Adenovirus with a Capsid-Incorporated Dual-Imaging Moiety for Monitoring Virotherapy in Ovarian Cancer

Directory of Open Access Journals (Sweden)

Kristopher J. Kimball

2009-09-01

Full Text Available We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for non-invasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk and monomeric red fluorescent protein 1 (mRFP1 into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis.

Science.gov (United States)

Bi, Lianxiang; Wacker, Bradley K; Bueren, Emma; Ham, Ervin; Dronadula, Nagadhara; Dichek, David A

2017-12-15

Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate-yet significant-pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

Anti-tumor effect of adenovirus-mediated suicide gene therapy under control of tumor-specific and radio-inducible chimeric promoter in combination with γ-ray irradiation in vivo

International Nuclear Information System (INIS)

Sun Wenjie; Yu Haijun; Xiongjie; Xu Yu; Liao Zhengkai; Zhou Fuxiang; Xie Conghua; Zhou Yunfeng

2011-01-01

Objective: To detect the selective inhibitory effects of irradiation plus adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) suicide gene system using tumor-specific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse. Methods: Recombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radio-inducible CArG elements was constructed. A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA. The change of tumor volume and tumor growth inhibiting rate, the survival time of nude mice, as well as histopathology of xenograft tumor and normal tissues were evaluated. Results: Thirty one days after the treatment, the relative tumor volumes in the negative, adenovirus therapy, irradiation, and combination groups were 49.23±4.55, 27.71±7.74, 28.53±10.48 and 11.58±3.23, respectively.There was a significantly statistical difference among them (F=16.288, P<0.01).The inhibition effect in the combination group was strongest as compared with that in other groups, and its inhibition ratio was 76.5%. The survival period extended to 43 d in the combination group, which showed a significantly difference with that in the control group (χ 2 =18.307, P<0.01). The area of tumors necrosis in the combination group was larger than that in the other groups, and the normal tissues showed no treatment-related toxic effect in all groups. However, multiple hepatocellular carcinoma metastases were observed in the liver in the control group, there were a few metastases in the monotherapy groups and no metastasis in the combination group. Conclusions: Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong

Up-regulation of integrin β3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy

International Nuclear Information System (INIS)

Egami, Takuya; Ohuchida, Kenoki; Yasui, Takaharu; Onimaru, Manabu; Toma, Hiroki; Sato, Norihiro; Tanaka, Masao; Mizumoto, Kazuhiro; Matsumoto, Kunio

2009-01-01

Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P<0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. (author)

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

National Research Council Canada - National Science Library

Curiel, David T; Siegal, Gene; Wang, Minghui

2007-01-01

...) to achieve efficient and selective gene transfer to target tumor cells. Proposed herein is a strategy to modify one candidate vector, recombinant adenovirus, such that it embodies the requisite properties of efficacy and specificity...

Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

International Nuclear Information System (INIS)

Cai Xuwei; Fu Xiaolong; Yang Jian; Song Houyan

2005-01-01

Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- X TM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-X TM genome, which was transformed into E. coli to get recombinant Adeno-X TM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 10 11 TCID 50 /ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-X TM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

Fiber mediated receptor masking in non-infected bystander cells restricts adenovirus cell killing effect but promotes adenovirus host co-existence.

Directory of Open Access Journals (Sweden)

Johan Rebetz

Full Text Available The basic concept of conditionally replicating adenoviruses (CRAD as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI, and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

Directory of Open Access Journals (Sweden)

Daniel C Farley

Full Text Available It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02. VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k.

Science.gov (United States)

Condezo, Gabriela N; Marabini, Roberto; Ayora, Silvia; Carazo, José M; Alba, Raúl; Chillón, Miguel; San Martín, Carmen

2015-09-01

Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the

Adenovirus Vector E4 Gene Regulates Connexin 40 and 43 Expression in Endothelial Cells via PKA and PI3K Signal Pathways

Science.gov (United States)

Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K.; Vincent, Loïc; Hackett, Neil R.; Wang, Shiyang; Young, Lauren M.; Hempstead, Barbara; Crystal, Ronald G.; Rafii, Shahin

2010-01-01

Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4−, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intratracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways. PMID:15831817

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

Science.gov (United States)

Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

2013-10-17

Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

International Nuclear Information System (INIS)

Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian

2007-01-01

The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10 8 PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization

STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

Directory of Open Access Journals (Sweden)

Karoly Toth

2015-08-01

Full Text Available Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as

DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

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Ilin Chuang

Full Text Available BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad. The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea, possibly related to immunization, was severe (Grade 3, preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27% were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102 and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270 and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019. Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%. Protection

Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

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Emma-Jo Hayton

Full Text Available HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern.These data demonstrate safety and good tolerability of the pSG2

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor (BDNF) tohuman umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) promotescrush-injured rat sciatic nerve regeneration.

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Hei, Wei-Hong; Almansoori, Akram A; Sung, Mi-Ae; Ju, Kyung-Won; Seo, Nari; Lee, Sung-Ho; Kim, Bong-Ju; Kim, Soung-Min; Jahng, Jeong Won; He, Hong; Lee, Jong-Ho

2017-03-16

This study was designed toinvestigate the efficacy of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in a rat sciatic nerve crush injury model. BDNF protein and mRNA expression after infection was checked through an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Male Sprague-Dawley rats (200-250g, 6 weeks old) were distributed into threegroups (n=20 each): the control group, UCB-MSC group, and BDNF-adenovirus infected UCB-MSC (BDNF-Ad+UCB-MSC) group. UCB-MSCs (1×10 6 cells/10μl/rat) or BDNF-Ad+UCB-MSCs (1×10 6 cells/10μl/rat)were transplantedinto the rats at the crush site immediately after sciatic nerve injury. Cell tracking was done with PKH26-labeled UCB-MSCs and BDNF-Ad+UCB-MSCs (1×10 6 cells/10μl/rat). The rats were monitored for 4 weeks post-surgery. Results showed that expression of BDNF at both the protein and mRNA levels was higher inthe BDNF-Ad+UCB-MSC group compared to theUCB-MSC group in vitro.Moreover, BDNF mRNA expression was higher in both UCB-MSC group and BDNF-Ad+ UCB-MSC group compared tothe control group, and BDNF mRNA expression in theBDNF-Ad+UCB-MSC group was higher than inboth other groups 5days after surgeryin vivo. Labeled neurons in the dorsal root ganglia (DRG), axon counts, axon density, and sciatic function index were significantly increased in the UCB-MSC and BDNF-Ad+ UCB-MSCgroupscompared to the controlgroup four weeksaftercell transplantation. Importantly,the BDNF-Ad+UCB-MSCgroup exhibited more peripheral nerve regeneration than the other two groups.Our results indicate thatboth UCB-MSCs and BDNF-Ad+UCB-MSCscan improve rat sciatic nerve regeneration, with BDNF-Ad+UCB-MSCsshowing a greater effectthan UCB-MSCs. Copyright © 2017 Elsevier B.V. All rights reserved.

A molecular epidemiology survey of respiratory adenoviruses circulating in children residing in Southern Palestine.

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Lina Qurei

Full Text Available A molecular epidemiology survey was performed in order to establish and document the respiratory adenovirus pathogen profiles among children in Southern Palestine. Three hundred and thirty-eight hospitalized pediatric cases with adenovirus-associated respiratory tract infections were analyzed. Forty four cases out of the 338 were evaluated in more detail for the adenoviruses types present. All of the children resided in Southern Palestine, that is, in city, village and refugee camp environments within the districts of Hebron and Bethlehem. Human adenoviruses circulated throughout 2005-2010, with major outbreaks occurring in the spring months. A larger percent of the children diagnosed with adenoviral infections were male infants. DNA sequence analysis of the hexon genes from 44 samples revealed that several distinct adenovirus types circulated in the region; these were HAdV-C1, HAdV-C2, HAdV-B3 and HAdV-C5. However, not all of these types were detected within each year. This is the first study ever conducted in Palestine of the genetic epidemiology of respiratory adenovirus infections.

Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus

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Amr Matoq

2016-01-01

Full Text Available Adenoviruses are a common cause of respiratory infection, pharyngitis, and conjunctivitis in infants and young children. They are known to cause hepatitis and liver failure in immunocompromised patients; they are a rare cause of hepatitis in immunocompetent patients and have been known to cause fulminant hepatic failure. We present a 23-month-old immunocompetent infant who presented with acute noncholestatic hepatitis, hypoalbuminemia, generalized anasarca, and pancytopenia secondary to adenovirus infection.

NF-κB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL

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Karacay Bahri

2010-10-01

Full Text Available Abstract Background Lung cancer causes the highest rate of cancer-related deaths both in men and women. As many current treatment modalities are inadequate in increasing patient survival, new therapeutic strategies are required. TNF-related apoptosis-inducing ligand (TRAIL selectively induces apoptosis in tumor cells but not in normal cells, prompting its current evaluation in a number of clinical trials. The successful therapeutic employment of TRAIL is restricted by the fact that many tumor cells are resistant to TRAIL. The goal of the present study was to test a novel combinatorial gene therapy modality involving adenoviral delivery of TRAIL (Ad5hTRAIL and IKK inhibition (AdIKKβKA to overcome TRAIL resistance in lung cancer cells. Methods Fluorescent microscopy and flow cytometry were used to detect optimum doses of adenovirus vectors to transduce lung cancer cells. Cell viability was assessed via a live/dead cell viability assay. Luciferase assays were employed to monitor cellular NF-κB activity. Apoptosis was confirmed using Annexin V binding. Results Neither Ad5hTRAIL nor AdIKKβKA infection alone induced apoptosis in A549 lung cancer cells, but the combined use of Ad5hTRAIL and AdIKKβKA significantly increased the amount of A549 apoptosis. Luciferase assays demonstrated that both endogenous and TRAIL-induced NF-κB activity was down-regulated by AdIKKβKA expression. Conclusions Combination treatment with Ad5hTRAIL and AdIKKβKA induced significant apoptosis of TRAIL-resistant A549 cells, suggesting that dual gene therapy strategy involving exogenous TRAIL gene expression with concurrent IKK inhibition may be a promising novel gene therapy modality to treat lung cancer.

NF-κB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL

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Aydin, Cigdem; Sanlioglu, Ahter D; Bisgin, Atil; Yoldas, Burcak; Dertsiz, Levent; Karacay, Bahri; Griffith, Thomas S; Sanlioglu, Salih

2010-01-01

Lung cancer causes the highest rate of cancer-related deaths both in men and women. As many current treatment modalities are inadequate in increasing patient survival, new therapeutic strategies are required. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells but not in normal cells, prompting its current evaluation in a number of clinical trials. The successful therapeutic employment of TRAIL is restricted by the fact that many tumor cells are resistant to TRAIL. The goal of the present study was to test a novel combinatorial gene therapy modality involving adenoviral delivery of TRAIL (Ad5hTRAIL) and IKK inhibition (AdIKKβKA) to overcome TRAIL resistance in lung cancer cells. Fluorescent microscopy and flow cytometry were used to detect optimum doses of adenovirus vectors to transduce lung cancer cells. Cell viability was assessed via a live/dead cell viability assay. Luciferase assays were employed to monitor cellular NF-κB activity. Apoptosis was confirmed using Annexin V binding. Neither Ad5hTRAIL nor AdIKKβKA infection alone induced apoptosis in A549 lung cancer cells, but the combined use of Ad5hTRAIL and AdIKKβKA significantly increased the amount of A549 apoptosis. Luciferase assays demonstrated that both endogenous and TRAIL-induced NF-κB activity was down-regulated by AdIKKβKA expression. Combination treatment with Ad5hTRAIL and AdIKKβKA induced significant apoptosis of TRAIL-resistant A549 cells, suggesting that dual gene therapy strategy involving exogenous TRAIL gene expression with concurrent IKK inhibition may be a promising novel gene therapy modality to treat lung cancer

Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

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Cheetham, B F; Shaw, D C; Bellett, A J

1982-01-01

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation. PMID:7177112

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

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Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy

2013-01-01

to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance...... is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription...

Genetic manipulation of endosymbionts to control vector and vector borne diseases

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Jay Prakash Gupta

Full Text Available Vector borne diseases (VBD are on the rise because of failure of the existing methods of control of vector and vector borne diseases and the climate change. A steep rise of VBDs are due to several factors like selection of insecticide resistant vector population, drug resistant parasite population and lack of effective vaccines against the VBDs. Environmental pollution, public health hazard and insecticide resistant vector population indicate that the insecticides are no longer a sustainable control method of vector and vector-borne diseases. Amongst the various alternative control strategies, symbiont based approach utilizing endosymbionts of arthropod vectors could be explored to control the vector and vector borne diseases. The endosymbiont population of arthropod vectors could be exploited in different ways viz., as a chemotherapeutic target, vaccine target for the control of vectors. Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting arthropods may serve as a powerful approach to control certain arthropod-borne diseases. Genetic transformation of symbiotic bacteria of the arthropod vector to alter the vector’s ability to transmit pathogen is an alternative means of blocking the transmission of VBDs. In Indian scenario, where dengue, chikungunya, malaria and filariosis are prevalent, paratransgenic based approach can be used effectively. [Vet World 2012; 5(9.000: 571-576

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

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Leopold, Philip L; Wendland, Rebecca L; Vincent, Theresa; Crystal, Ronald G

2006-10-01

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

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Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics ...

Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

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Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

2017-12-01

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

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Hu Shunying; Chen Yundai; Li Libing; Chen Jinlong; Wu Bin; Zhou, Xiao; Zhi Guang; Li Qingfang; Wang Rongliang; Duan Haifeng; Guo Zikuan; Yang Yuefeng; Xiao Fengjun; Wang Hua; Wang Lisheng

2009-01-01

Purpose: Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. Methods and Materials: Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. Results: Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 ± 9.01 vs. 41.94 ± 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 ± 1303 vs. 1667 ± 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 ± 1084 vs. 1566 ± 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. Conclusion: Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

Long-circulating DNA lipid nanocapsules as new vector for passive tumor targeting.

OpenAIRE

Morille , Marie; Montier , Tristan; Legras , Pierre; Carmoy , Nathalie; Brodin , Priscille; Pitard , Bruno; Benoît , Jean-Pierre; Passirani , Catherine

2010-01-01

International audience; Systemic gene delivery systems are needed for therapeutic application to organs that are inaccessible by percutaneous injection. Currently, the main objective is the development of a stable and non-toxic vector that can encapsulate and deliver foreign genetic material to target cells. To this end, DNA, complexed with cationic lipids i.e. DOTAP/DOPE, was encapsulated into lipid nanocapsules (LNCs) leading to the formation of stable nanocarriers (DNA LNCs) with a size in...

A simple negative selection method to identify adenovirus recombinants using colony PCR

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Yongliang Zhao

2014-01-01

Conclusions: The negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging.

The search for adenovirus 14 in children in Houston, Texas.

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Laham, Federico R; Jewell, Alan M; Schoonover, Shauna L; Demmler, Gail J; Piedra, Pedro A

2008-07-01

Adenovirus (Ad)14 has recently emerged in the United States causing outbreaks of severe respiratory disease. To determine if Ad14 circulated in Houston, Texas, during the same time as an outbreak in military recruits in nearby San Antonio, 215 pediatric adenovirus isolates were serotyped using microneutralization. None were Ad14; Ad1, Ad2, and Ad3 were the most common identified serotypes.

Respiratory adenovirus-like infection in a rose-ringed parakeet (Psittacula krameri).

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Desmidt, M; Ducatelle, R; Uyttebroek, E; Charlier, G; Hoorens, J

1991-01-01

Intranuclear inclusions were observed under light microscopy in the bronchial epithelial cells of a recently purchased female rose-ringed parakeet that died of chlamydiosis. Transmission electron microscopy revealed the presence of numerous particles of adenovirus morphology. A latent adenovirus infection may have become more severe following chlamydiosis and the stress of handling.

Genetic and Molecular Epidemiological Characterization of a Novel Adenovirus in Antarctic Penguins Collected between 2008 and 2013.

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Sook-Young Lee

Full Text Available Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica and two Gentoo penguins (Pygoscelis papua. The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630-24,662 bp and 35.5-35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9-39.3% (amino acid, 32.1-47.9% in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3 in phylogenetic analysis. During the 2008-2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%, and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%. Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population.

A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

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Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

2016-09-01

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

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HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

2001-08-01

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis

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Lianxiang Bi

2017-12-01

Full Text Available Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

International Nuclear Information System (INIS)

Wei, Na; Fan, Jun Kai; Gu, Jin Fa; He, Ling Feng; Tang, Wen Hao; Cao, Xin; Liu, Xin Yuan

2009-01-01

Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.

Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

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Xishuang Liu

2011-01-01

Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

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Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

Adenovirus serotype 7 associated with a severe lower respiratory tract disease outbreak in infants in Shaanxi Province, China

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Xu Wenbo

2011-01-01

Full Text Available Abstract Background Pneumonia caused by adenovirus infection is usually severe especially with adenovirus serotype 7 commonly associated with lower respiratory tract disease outbreaks. We reported an outbreak of 70 cases of severe pneumonia with one death of infants in Shaanxi Province, China. Sampling showed adenovirus 7 (Ad7 as the primary pathogen with some co-infections. Results Two strains of adenovirus and two strains of enterovirus were isolated, the 21 pharynx swabs showed 14 positive amplifications for adenovirus; three co-infections with respiratory syncytial virus, two positive for rhinovirus, one positive for parainfluenza 3, and four negative. Adenovirus typing showed nine of the nine adenovirus positive samples were HAdV-7, three were HAdV-3 and two were too weak to perform sequencing. The entire hexon gene of adenovirus was sequenced and analyzed for the two adenovirus serotype 7 isolates, showing the nucleic acid homology was 99.8% between the two strains and 99.5% compared to the reference strain HAdV-7 (GenBank accession number AY769946. For the 21 acute phase serum samples from the 21 patients, six samples had positives results for ELISA detection of HAdV IgA, and the neutralization titers of the convalescent-phase samples were four times higher than those of the acute-phase samples in nine pairs. Conclusions We concluded adenovirus was the viral pathogen, primarily HAdV-7, with some co-infections responsible for the outbreak. This is the first report of an infant pneumonia outbreak caused by adenovirus serotype 7 in Shaanxi Province, China.

Simian virus 40 vectors for pulmonary gene therapy

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Oppenheim Ariella

2007-10-01

Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

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Xingui Tian

2015-10-01

Full Text Available Human adenovirus type 55 (HAdV55 is a newly identified re-emergent acute respiratory disease (ARD pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152, A55R2 (residues 179 to 187, A55R4 (residues 247 to 259 and A55R7 (residues 429 to 443, were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA and neutralization tests (NT. Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3 and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis.

Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

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Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

2015-01-01

Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

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Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Adenovirus urethritis and concurrent conjunctivitis: a case series and review of the literature.

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Liddle, Olivia Louise; Samuel, Mannampallil Itty; Sudhanva, Malur; Ellis, Joanna; Taylor, Chris

2015-03-01

We present eight cases and review the literature of concurrent urethritis and conjunctivitis where adenovirus was identified as the causative pathogen. The focus of this review concerns the identification of specific sexual practices, symptoms, signs and any serotypes that seem more commonly associated with such adenovirus infections. We discuss the seasonality of adenovirus infection and provide practical advice for clinicians to give to the patient. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Screening for adenoviruses in haematological neoplasia: High prevalence in mantle cell lymphoma.

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Kosulin, Karin; Rauch, Margit; Ambros, Peter F; Pötschger, Ulrike; Chott, Andreas; Jäger, Ulrich; Drach, Johannes; Nader, Alexander; Lion, Thomas

2014-02-01

Human adenoviruses possess oncogenic capacity which is well documented in mammalian animal models, but their possible implication in human malignancy has remained enigmatic. Following primary infection, adenoviruses can persist in a latent state in lymphocytes where the virus is apparently able to evade immune surveillance. In the present study, we have employed a broad-spectrum adenovirus polymerase chain reaction (PCR) assay to systematically screen more than 200 diagnostic specimens of different lymphoid malignancies including acute lymphocytic leukaemia (n=50), chronic lymphocytic leukaemia (n=50), various types of malignant lymphoma (n=100) and multiple myeloma (n=11) for the presence of adenoviral sequences. While most entities analysed revealed negative findings in virtually all specimens tested, adenoviral DNA was detected in 15/36 (42%) mantle cell lymphomas investigated. The most prevalent adenoviral species detected was C, and less commonly B. Adenovirus-positive findings in patients with mantle cell lymphoma were made at different sites including bone marrow (n=7), intestine (n=5), lymph nodes (n=2) and tonsillar tissue (n=1). The presence of adenoviral sequences identified by PCR was confirmed in individual cells by fluorescence in-situ hybridisation (FISH). The frequent observation of adenoviruses in mantle cell lymphoma is intriguings, and raises questions about their possible involvement in the pathogenesis of this lymphoid malignancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Derivation of a triple mosaic adenovirus for cancer gene therapy.

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Yizhe Tang

2009-12-01

Full Text Available A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK, the herpes simplex virus type 1 (HSV-1 thymidine kinase (TK, and the monomeric red fluorescent protein (mRFP1 as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX. In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors.

Experimental cross-species infection of common marmosets by titi monkey adenovirus.

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Guixia Yu

Full Text Available Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV, as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus. Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

Structure of a Reptilian Adenovirus Reveals a Phage Tailspike Fold Stabilizing a Vertebrate Virus Capsid.

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Menéndez-Conejero, Rosa; Nguyen, Thanh H; Singh, Abhimanyu K; Condezo, Gabriela N; Marschang, Rachel E; van Raaij, Mark J; San Martín, Carmen

2017-10-03

Although non-human adenoviruses (AdVs) might offer solutions to problems posed by human AdVs as therapeutic vectors, little is known about their basic biology. In particular, there are no structural studies on the complete virion of any AdV with a non-mammalian host. We combine mass spectrometry, cryo-electron microscopy, and protein crystallography to characterize the composition and structure of a snake AdV (SnAdV-1, Atadenovirus genus). SnAdV-1 particles contain the genus-specific proteins LH3, p32k, and LH2, a previously unrecognized structural component. Remarkably, the cementing protein LH3 has a trimeric β helix fold typical of bacteriophage host attachment proteins. The organization of minor coat proteins differs from that in human AdVs, correlating with higher thermostability in SnAdV-1. These findings add a new piece to the intriguing puzzle of virus evolution, hint at the use of cell entry pathways different from those in human AdVs, and will help development of new, thermostable SnAdV-1-based vectors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene therapy in the inner ear using adenovirus vectors.

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Husseman, Jacob; Raphael, Yehoash

2009-01-01

Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression. Copyright (c) 2009 S. Karger AG, Basel.

Deaths from Adenovirus in the US Military

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Dr. Joel Gaydos, science advisor for the Armed Forces Health Surveillance Center, and Dr. Robert Potter, a research associate for the Armed Forces Medical Examiner System, discuss deaths from adenovirus in the US military.

An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma.

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Zhang, Yan; Fang, Lin; Zhang, Quan'an; Zheng, Qin; Tong, Jinlong; Fu, Xiaohui; Jiang, Xiaoqing; Su, Changqing; Zheng, Junnian

2013-06-01

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effects of the deletion of early region 4 (E4 open reading frame 1 (orf1, orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

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Michael A Thomas

Full Text Available The global health burden engendered by human immunodeficiency virus (HIV-induced acquired immunodeficiency syndrome (AIDS is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1 through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

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Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a

Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

International Nuclear Information System (INIS)

Steenbergh, P.H.

1979-01-01

The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

OpenAIRE

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.; Nettelbeck, Dirk M.

2010-01-01

Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show t...

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

Science.gov (United States)

Molino, Yves; David, Marion; Varini, Karine; Jabès, Françoise; Gaudin, Nicolas; Fortoul, Aude; Bakloul, Karima; Masse, Maxime; Bernard, Anne; Drobecq, Lucile; Lécorché, Pascaline; Temsamani, Jamal; Jacquot, Guillaume; Khrestchatisky, Michel

2017-05-01

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. © FASEB.

Construction of lentiviral shRNA expression vector targeting ...

African Journals Online (AJOL)

DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and ...

In silico models for predicting vector control chemicals targeting Aedes aegypti

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Devillers, J.; Lagneau, C.; Lattes, A.; Garrigues, J.C.; Clémenté, M.M.; Yébakima, A.

2014-01-01

Human arboviral diseases have emerged or re-emerged in numerous countries worldwide due to a number of factors including the lack of progress in vaccine development, lack of drugs, insecticide resistance in mosquitoes, climate changes, societal behaviours, and economical constraints. Thus, Aedes aegypti is the main vector of the yellow fever and dengue fever flaviviruses and is also responsible for several recent outbreaks of the chikungunya alphavirus. As for the other mosquito species, the A. aegypti control relies heavily on the use of insecticides. However, because of increasing resistance to the different families of insecticides, reduction of Aedes populations is becoming increasingly difficult. Despite the unquestionable utility of insecticides in fighting mosquito populations, there are very few new insecticides developed and commercialized for vector control. This is because the high cost of the discovery of an insecticide is not counterbalanced by the ‘low profitability’ of the vector control market. Fortunately, the use of quantitative structure–activity relationship (QSAR) modelling allows the reduction of time and cost in the discovery of new chemical structures potentially active against mosquitoes. In this context, the goal of the present study was to review all the existing QSAR models on A. aegypti. The homology and pharmacophore models were also reviewed. Specific attention was paid to show the variety of targets investigated in Aedes in relation to the physiology and ecology of the mosquito as well as the diversity of the chemical structures which have been proposed, encompassing man-made and natural substances. PMID:25275884

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

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Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles▿

OpenAIRE

Chung, Sang-Hyuk; Frese, Kristopher K.; Weiss, Robert S.; Prasad, B. V. Venkataram; Javier, Ronald T.

2007-01-01

Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, inc...

Adenovirus-dependent changes in cell membrane permeability: role of Na/sup +/, K/sup +/-ATPase

Energy Technology Data Exchange (ETDEWEB)

Seth, P.; Pastan, I.; Willingham, M.C.

1987-03-01

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K/sup +/-containing medium, maximum inhibition of release was obtained by 10/sup -5/ M ouabain and half-maximal inhibition was achieved by about 0.5 x 10/sup -6/ M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K/sup +/, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K/sup +/ about 50% was released. This activation by K/sup +/ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na/sup +/, K/sup +/-ATPase in KB cells, with maximum activation at 50..mu..g of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na/sup +/, K/sup +/, ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K/sup +/ was present in the medium. Under the conditions used, K/sup +/ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na/sup +/, K/sup +/-ATPase activity.

Sequential and Simultaneous Applications of UV and Chlorine for Adenovirus Inactivation.

Science.gov (United States)

Rattanakul, Surapong; Oguma, Kumiko; Takizawa, Satoshi

2015-09-01

Adenoviruses are water-borne human pathogens with high resistance to UV disinfection. Combination of UV treatment and chlorination could be an effective approach to deal with adenoviruses. In this study, human adenovirus 5 (HAdV-5) was challenged in a bench-scale experiment by separate applications of UV or chlorine and by combined applications of UV and chlorine in either a sequential or simultaneous manner. The treated samples were then propagated in human lung carcinoma epithelial cells to quantify the log inactivation of HAdV-5. When the processes were separate, a fluence of 100 mJ/cm(2) and a CT value of 0.02 mg min/L were required to achieve 2 log inactivation of HAdV-5 by UV disinfection and chlorination, respectively. Interestingly, synergistic effects on the HAdV-5 inactivation rates were found in the sequential process of chlorine followed by UV (Cl2-UV) (p simultaneous application of UV/Cl2. This implies that a pretreatment with chlorine may increase the sensitivity of the virus to the subsequent UV disinfection. In conclusion, this study suggests that the combined application of UV and chlorine could be an effective measure against adenoviruses as a multi-barrier approach in water disinfection.

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

Science.gov (United States)

Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

2015-01-01

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

Molecular design for recombinant adeno-associated virus (rAAV) vector production.

Science.gov (United States)

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

2018-02-01

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

Bipartite structure and functional independence of adenovirus type 5 packaging elements.

OpenAIRE

Schmid, S I; Hearing, P

1997-01-01

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements ...

Interspecies differences in virus uptake versus cardiac function of the coxsackievirus and adenovirus receptor.

NARCIS (Netherlands)

Freiberg, F.; Sauter, M.; Pinkert, S.; Govindarajan, T.; Kaldrack, J.; Thakkar, M.; Fechner, H.; Klingel, K.; Gotthardt, M.

2014-01-01

The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail

Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice.

Science.gov (United States)

Badamchi-Zadeh, Alexander; Tartaglia, Lawrence J; Abbink, Peter; Bricault, Christine A; Liu, Po-Ting; Boyd, Michael; Kirilova, Marinela; Mercado, Noe B; Nanayakkara, Ovini S; Vrbanac, Vladimir D; Tager, Andrew M; Larocca, Rafael A; Seaman, Michael S; Barouch, Dan H

2018-04-01

Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Copyright © 2018 Badamchi-Zadeh et al.

Adenoviral vector immunity: its implications and circumvention strategies.

Science.gov (United States)

Ahi, Yadvinder S; Bangari, Dinesh S; Mittal, Suresh K

2011-08-01

Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1

Czech Academy of Sciences Publication Activity Database

Carlisle, R. C.; Di, Y.; Cerny, A. M.; Sonnen, A. F. P.; Sim, R. B.; Green, N. K.; Šubr, Vladimír; Ulbrich, Karel; Gilbert, R. J. C.; Fisher, K. D.; Finberg, R. W.; Seymour, L. W.

2009-01-01

Roč. 113, č. 9 (2009), s. 1909-1918 ISSN 0006-4971 EU Projects: European Commission(XE) 512087 - GIANT Institutional research plan: CEZ:AV0Z40500505 Keywords : adenovirus * erythrocyte * complement receptor 1 Subject RIV: CD - Macromolecular Chemistry Impact factor: 10.555, year: 2009

HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

International Nuclear Information System (INIS)

Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig; Jameson, J. Larry

2008-01-01

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the β-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms

Probing deformed orbitals with vector A( vector e, e' N)B reactions

International Nuclear Information System (INIS)

Garrido, E.; Caballero, J.A.; Moya de Guerra, E.; Sarriguren, P.; Udias, J.M.

1995-01-01

We present results for response functions and asymmetries in the nuclear reactions 37 vector Ar( vector e, e' n) 36 Ar and 37 vector K( vector e,e' p) 36 Ar at quasifree kinematics. We compare PWIA results obtained using deformed HF wave functions with PWIA and DWIA results obtained assuming a spherical mean field. We show that the complex structure of the deformed orbitals can be probed by coincidence measurements with polarized beam and targets. ((orig.))

Oral Modeling of an Adenovirus-Based Quadrivalent Influenza Vaccine in Ferrets and Mice.

Science.gov (United States)

Scallan, Ciaran D; Lindbloom, Jonathan D; Tucker, Sean N

2016-06-01

Oral vaccines delivered as tablets offer a number of advantages over traditional parenteral-based vaccines including the ease of delivery, lack of needles, no need for trained medical personnel, and the ability to formulate into temperature-stable tablets. We have been evaluating an oral vaccine platform based on recombinant adenoviral vectors for the purpose of creating a prophylactic vaccine to prevent influenza, and have demonstrated vaccine efficacy in animal models and substantial immunogenicity in humans. These studies have evaluated monovalent vaccines to date. To protect against the major circulating A and B influenza strains, a multivalent influenza vaccine will be required. In this study, the immunogenicity of orally delivered monovalent, bivalent, trivalent, and quadrivalent vaccines was tested in ferrets and mice. The various vaccine combinations were tested by blending monovalent recombinant adenovirus vaccines, each expressing hemagglutinin from a single strain. Human tablet delivery was modeled in animals by oral gavage in mice and by endoscopic delivery in ferrets. We demonstrated minimal interference between the various vaccine vectors when used in combination and that the oral quadrivalent vaccine compared favorably to an approved trivalent inactivated vaccine. The quadrivalent vaccine presented here produced immune responses that we predict should be capable of providing protection against multiple influenza strains, and the platform should have applications to other multivalent vaccines. Vaxart, Inc.

Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging

International Nuclear Information System (INIS)

Maeda, Yasushi; Kimura, En; Uchida, Yuji; Nishida, Yasuto; Yamashita, Satoshi; Arima, Toshiyuki; Uchino, Makoto

2003-01-01

Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination

Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

DEFF Research Database (Denmark)

Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov

2008-01-01

Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion...... of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

Directory of Open Access Journals (Sweden)

Rizzo Nidia

2012-10-01

Full Text Available Abstract Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.. The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae. After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed in 970 study

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

Science.gov (United States)

Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

2012-10-30

In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

Immunizing Patients With Metastatic Melanoma Using Recombinant Adenoviruses Encoding MART-1 or gp100 Melanoma Antigens

Science.gov (United States)

Rosenberg, Steven A.; Zhai, Yifan; Yang, James C.; Schwartzentruber, Douglas J.; Hwu, Patrick; Marincola, Francesco M.; Topalian, Suzanne L.; Restifo, Nicholas P.; Seipp, Claudia A.; Einhorn, Jan H.; Roberts, Bruce; White, Donald E.

2008-01-01

Background: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. Methods: In phase I studies, 54 patients received escalating doses (between 107 and 1011 plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. Results: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. Conclusions: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens. PMID:9862627

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

Science.gov (United States)

Sha, Jian; Kirtley, Michelle L; Klages, Curtis; Erova, Tatiana E; Telepnev, Maxim; Ponnusamy, Duraisamy; Fitts, Eric C; Baze, Wallace B; Sivasubramani, Satheesh K; Lawrence, William S; Patrikeev, Igor; Peel, Jennifer E; Andersson, Jourdan A; Kozlova, Elena V; Tiner, Bethany L; Peterson, Johnny W; McWilliams, David; Patel, Snehal; Rothe, Eric; Motin, Vladimir L; Chopra, Ashok K

2016-07-01

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The E1A proteins of all six human adenovirus subgroups target the p300/CBP acetyltransferases and the SAGA transcriptional regulatory complex

International Nuclear Information System (INIS)

Shuen, Michael; Avvakumov, Nikita; Torchia, Joe; Mymryk, Joe S.

2003-01-01

The N-terminal/conserved region 1 (CR1) portion of the human adenovirus (Ad) 5 E1A protein was previously shown to inhibit growth in the simple eukaryote Saccharomyces cerevisiae. We now demonstrate that the corresponding regions of the E1A proteins of Ad3,-4,-9,-12, and -40, which represent the remaining five Ad subgroups, also inhibit yeast growth. These results suggest that the E1A proteins of all six human Ad subgroups share a common cellular target(s) conserved in yeast. Growth inhibition induced by either full-length or the N-terminal/CR1 portion of Ad5 E1A was relieved by coexpression of the E1A binding portions of the mammalian p300, CBP, and pCAF acetyltransferases. Similarly, growth inhibition by the N-terminal/CR1 portions of the other Ad E1A proteins was suppressed by expression of the same regions of CBP or pCAF known to bind Ad5 E1A. The physical interaction of each of the different Ad E1A proteins with CBP, p300, and pCAF was confirmed in vitro. Furthermore, deletion of the gene encoding yGcn5, the yeast homolog of pCAF and a subunit of the SAGA transcriptional regulatory complex, restored growth in yeast expressing each of the different Ad E1A proteins. This indicates that the SAGA complex is a conserved target of all Ad E1A proteins. Our results demonstrate for the first time that the p300, CBP, and pCAF acetyltransferases are common targets for the E1A proteins of all six human Ad subgroups, highlighting the importance of these interactions for E1A function

A technique for measurement of vector and tensor polarization in solid spin one polarized targets

International Nuclear Information System (INIS)

Kielhorn, W.F.

1991-06-01

Vector and tensor polarizations are explicitly defined and used to characterize the polarization states of spin one polarized targets, and a technique for extracting these polarizations from nuclear magnetic resonance (NMR) data is developed. This technique is independent of assumptions about spin temperature, but assumes the target's crystal structure induces a quadrupole interaction with the spin one particles. Analysis of the NMR signals involves a computer curve fitting algorithm implemented with a fast Fourier transform method which speeds and simplifies curve fitting algorithms used previously. For accurate curve fitting, the NMR electronic circuit must be modeled by the fitting algorithm. Details of a circuit, its model, and data collected from this circuit are given for a solid deuterated ammonia target. 37 refs., 19 figs., 3 tabs

characterisation of gastro- enteritis-associated adenoviruses in ...

African Journals Online (AJOL)

Objective. To analyse adenovirus (Ad) numbers and types associated with paediatric gastro-enteritis in South Africa. Setting. Gauteng, 1994-1996. Metfwds. A total of 234 paediatric diarrhoeal stool samples were screened for Ad using commercial enzyme-linked. iInmunosorbent assays (EUSAs). Adenoviral isolates were.

Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

International Nuclear Information System (INIS)

Rhee, Juong G.; Li, Daqing; Suntharalingam, Mohan; Guo Chuanfa; O'Malley, Bert W.; Carney, James P.

2007-01-01

Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers

Prolonged peritoneal gene expression using a helper-dependent adenovirus.

Science.gov (United States)

Liu, Limin; Shi, Chang-Xin; Ghayur, Ayesha; Zhang, Claire; Su, Je Yen; Hoff, Catherine M; Margetts, Peter J

2009-01-01

Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis. The causes of EPS are not well defined and are likely multifactorial. A suitable animal model would facilitate research into the pathophysiology and treatment of EPS. We developed a helper-dependent adenovirus that expresses both green fluorescent protein (GFP) and active transforming growth factor-beta (TGF-beta1; HDAdTGF-beta1). Mice were administered HDAdTGF-beta1 via intraperitoneal injection and the response was compared with mice administered either first-generation adenovirus expressing TGF-beta1 (AdTGF-beta1) or control adenovirus (AdGFP). HDAdTGF-beta1-treated mice continued to express the GFP reporter transgene to day 74, the end of the observation period. Transgene expression lasted less than 28 days in the animals treated with first-generation adenoviruses. Animals treated with first-generation AdTGF-beta1 demonstrated submesothelial thickening and angiogenesis at day 7, with almost complete resolution by day 28. The HDAdTGF-beta1-treated mice demonstrated progressive peritoneal fibrosis with adhesion formation and encapsulation of bowels. Weight gain was significantly reduced in animals treated with HDAdTGF-beta1 compared to both the control-treated animals and the AdTGF-beta1-treated animals. Inflammation was not a major component of the fibroproliferative response. Peritoneal administration of a first-generation AdTGF-beta1 leads to transient gene expression, resulting in a resolving fibrotic response and histology similar to that seen in simple peritoneal sclerosis. Prolonged TGF-beta1 expression induced by the helper-dependent HDAdTGF-beta1 led to changes in peritoneal morphology resembling EPS. This suggests that TGF-beta1 may be a contributing factor in both simple peritoneal sclerosis and EPS. This model will be useful for elucidation of the mechanism of EPS and evaluation of potential treatment.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

OpenAIRE

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice...

Thin-section computed tomography findings in 104 immunocompetent patients with adenovirus pneumonia.

Science.gov (United States)

Park, Chan Kue; Kwon, Hoon; Park, Ji Young

2017-08-01

Background To date, there has been no computed tomography (CT) evaluation of adenovirus pneumonia in a large number of immunocompetent patients. Purpose To describe the thin-section CT findings of immunocompetent patients with adenovirus pneumonia. Material and Methods We prospectively enrolled 104 patients with adenovirus pneumonia from a military hospital. CT scans of each patient were retrospectively and independently assessed by two radiologists for the presence of abnormalities, laterality and zonal predominance of the parenchymal abnormalities, and dominant imaging patterns and their anatomic distributions. Results CT findings included consolidation (n = 92), ground-glass opacity (GGO; n = 82), septal thickening (n = 34), nodules (n = 46), bronchial wall thickening (n = 32), pleural effusion (n = 16), and lymphadenopathy (n = 3). Eighty-four patients (81%) exhibited unilateral parenchymal abnormalities and 57 (57%) exhibited lower lung zone abnormalities. The most frequently dominant CT pattern was consolidation with surrounding GGO (n = 50), with subpleural (70%) and peribronchovascular (94%) distributions. Consolidation-the second-most common pattern (n = 33)-also exhibited subpleural (79%) and peribronchovascular (97%) distributions. The dominant nodule pattern (n = 14) exhibited mixed (64%) and peribronchovascular (100%) distributions. A dominant GGO pattern was only observed in four patients; none had central distribution. Conclusion Although the manifestations of adenovirus pneumonia on CT are varied, we found the most frequent pattern was consolidation with or without surrounding GGO, with subpleural and peribronchovascular distributions. Parenchymal abnormalities were predominantly unilateral and located in the lower lung zone. If dominant consolidation findings are present in immunocompetent patients during the early stages, adenovirus pneumonia should be considered.

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

DEFF Research Database (Denmark)

Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

2010-01-01

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic...

Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes

International Nuclear Information System (INIS)

Hyypiae, T.

1985-01-01

The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an enzyme immunoassay for adenovirus hexon antigen which was used as a reference test. Sixteen specimens positive by the enzyme immunoassay also were positive in the two nucleic acid hybridization tests, and the remaining eight specimens were negative in all of the tests. The results indicate that nucleid acid hybridization tests with both radioactive and nonradioactive probes can be used for diagnosis of microbial infections

Adenovirus Infection in Children with Diarrhea Disease in ...

African Journals Online (AJOL)

Ad40) and type 41(Ad41), can cause acute and severe diarrhea in young children worldwide. This study was conducted to delineate the epidemiological features of adenoviruses identified in children with gastroenteritis in Northwestern Nigeria.

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

Science.gov (United States)

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

Directory of Open Access Journals (Sweden)

E. Gout

2010-01-01

Full Text Available Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

Science.gov (United States)

Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

2018-01-01

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

Directory of Open Access Journals (Sweden)

Samuel Campbell

2018-04-01

Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control.

Science.gov (United States)

Peterson, Jennifer K; Bartsch, Sarah M; Lee, Bruce Y; Dobson, Andrew P

2015-10-22

Chagas disease (caused by Trypanosoma cruzi) is the most important neglected tropical disease (NTD) in Latin America, infecting an estimated 5.7 million people in the 21 countries where it is endemic. It is one of the NTDs targeted for control and elimination by the 2020 London Declaration goals, with the first goal being to interrupt intra-domiciliary vector-borne T. cruzi transmission. A key question in domestic T. cruzi transmission is the role that synanthropic animals play in T. cruzi transmission to humans. Here, we ask, (1) do synanthropic animals need to be targeted in Chagas disease prevention policies?, and (2) how does the presence of animals affect the efficacy of vector control? We developed a simple mathematical model to simulate domestic vector-borne T. cruzi transmission and to specifically examine the interaction between the presence of synanthropic animals and effects of vector control. We used the model to explore how the interactions between triatomine bugs, humans and animals impact the number and proportion of T. cruzi-infected bugs and humans. We then examined how T. cruzi dynamics change when control measures targeting vector abundance are introduced into the system. We found that the presence of synanthropic animals slows the speed of T. cruzi transmission to humans, and increases the sensitivity of T. cruzi transmission dynamics to vector control measures at comparable triatomine carrying capacities. However, T. cruzi transmission is amplified when triatomine carrying capacity increases with the abundance of syntathoropic hosts. Our results suggest that in domestic T. cruzi transmission scenarios where no vector control measures are in place, a reduction in synanthropic animals may slow T. cruzi transmission to humans, but it would not completely eliminate transmission. To reach the 2020 goal of interrupting intra-domiciliary T. cruzi transmission, it is critical to target vector populations. Additionally, where vector control measures

A technique for measurement of vector and tensor polarization in solid spin one polarized targets

Energy Technology Data Exchange (ETDEWEB)

Kielhorn, W.F.

1991-06-01

Vector and tensor polarizations are explicitly defined and used to characterize the polarization states of spin one polarized targets, and a technique for extracting these polarizations from nuclear magnetic resonance (NMR) data is developed. This technique is independent of assumptions about spin temperature, but assumes the target's crystal structure induces a quadrupole interaction with the spin one particles. Analysis of the NMR signals involves a computer curve fitting algorithm implemented with a fast Fourier transform method which speeds and simplifies curve fitting algorithms used previously. For accurate curve fitting, the NMR electronic circuit must be modeled by the fitting algorithm. Details of a circuit, its model, and data collected from this circuit are given for a solid deuterated ammonia target. 37 refs., 19 figs., 3 tabs.

The complete nucleotide sequence, genome organization, and origin of human adenovirus type 11

International Nuclear Information System (INIS)

Stone, Daniel; Furthmann, Anne; Sandig, Volker; Lieber, Andre

2003-01-01

The complete DNA sequence and transcription map of human adenovirus type 11 are reported here. This is the first published sequence for a subgenera B human adenovirus and demonstrates a genome organization highly similar to those of other human adenoviruses. All of the genes from the early, intermediate, and late regions are present in the expected locations of the genome for a human adenovirus. The genome size is 34,794 bp in length and has a GC content of 48.9%. Sequence alignment with genomes of groups A (Ad12), C (Ad5), D (Ad17), E (Simian adenovirus 25), and F (Ad40) revealed homologies of 64, 54, 68, 75, and 52%, respectively. Detailed genomic analysis demonstrated that Ads 11 and 35 are highly conserved in all areas except the hexon hypervariable regions and fiber. Similarly, comparison of Ad11 with subgroup E SAV25 revealed poor homology between fibers but high homology in proteins encoded by all other areas of the genome. We propose an evolutionary model in which functional viruses can be reconstituted following fiber substitution from one serotype to another. According to this model either the Ad11 genome is a derivative of Ad35, from which the fiber was substituted with Ad7, or the Ad35 genome is the product of a fiber substitution from Ad21 into the Ad11 genome. This model also provides a possible explanation for the origin of group E Ads, which are evolutionarily derived from a group C fiber substitution into a group B genome

Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses.

Science.gov (United States)

Wellehan, James F X; Johnson, April J; Harrach, Balázs; Benkö, Mária; Pessier, Allan P; Johnson, Calvin M; Garner, Michael M; Childress, April; Jacobson, Elliott R

2004-12-01

A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses.

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

Science.gov (United States)

Brown, Nolan; Song, Liujiang; Kollu, Nageswara R; Hirsch, Matthew L

2017-06-01

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the

Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

International Nuclear Information System (INIS)

Mu, Haixi; Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu; Li, Lili; Ren, Guosheng; Xu, Yongzhu; Zhou, Xiangyang; Xiang, Tingxiu

2015-01-01

Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer

Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

Energy Technology Data Exchange (ETDEWEB)

Mu, Haixi [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Li, Lili [Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute (Hong Kong); Ren, Guosheng [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Xu, Yongzhu [Chongqing Health Service Center, Chongqing 400020 (China); Zhou, Xiangyang [The Wistar Institute, Philadelphia, PA (United States); Xiang, Tingxiu, E-mail: xiangtx1@gmail.com [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China)

2015-03-15

Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

Directory of Open Access Journals (Sweden)

Jianfeng Zhang

2012-11-01

Full Text Available The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future.

Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

OpenAIRE

Wellehan, James F. X.; Johnson, April J.; Harrach, Balázs; Benkö, Mária; Pessier, Allan P.; Johnson, Calvin M.; Garner, Michael M.; Childress, April; Jacobson, Elliott R.

2004-01-01

A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phyloge...

Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a randomised, double-blind, placebo-controlled, phase 1 trial.

Science.gov (United States)

Zhu, Feng-Cai; Hou, Li-Hua; Li, Jing-Xin; Wu, Shi-Po; Liu, Pei; Zhang, Gui-Rong; Hu, Yue-Mei; Meng, Fan-Yue; Xu, Jun-Jie; Tang, Rong; Zhang, Jin-Long; Wang, Wen-Juan; Duan, Lei; Chu, Kai; Liang, Qi; Hu, Jia-Lei; Luo, Li; Zhu, Tao; Wang, Jun-Zhi; Chen, Wei

2015-06-06

Up to now, all tested Ebola virus vaccines have been based on the virus strain from the Zaire outbreak in 1976. We aimed to assess the safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain. We did this randomised, double-blind, placebo-controlled, phase 1 clinical trial at one site in Taizhou County, Jiangsu Province, China. Healthy adults (aged 18-60 years) were sequentially enrolled and randomly assigned (2:1), by computer-generated block randomisation (block size of six), to receive placebo, low-dose adenovirus type-5 vector-based Ebola vaccine, or high-dose vaccine. Randomisation was pre-stratified by dose group. All participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was occurrence of solicited adverse reactions within 7 days of vaccination. The primary immunogenicity endpoints were glycoprotein-specific antibody titres and T-cell responses at day 28 after the vaccination. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT02326194. Between Dec 28, 2014, and Jan 9, 2015, 120 participants were enrolled and randomly assigned to receive placebo (n=40), low-dose vaccine (n=40), or high-dose vaccine. Participants were followed up for 28 days. Overall, 82 (68%) participants reported at least one solicited adverse reaction within 7 days of vaccination (n=19 in the placebo group vs n=27 in the low-dose group vs n=36 in the high-dose group; p=0·0002). The most common reaction was mild pain at the injection site, which was reported in eight (20%) participants in the placebo group, 14 (35%) participants in the low-dose group, and 29 (73%) participants in the high-dose vaccine group (pvaccine groups at both day 14 (geometric mean titre 421·4 [95% CI 249·7-711·3] and 820·5 [598·9-1124·0], respectively; pday 28 (682·7 [424·3-1098·5] and 1305·7 [970·1-1757·2

Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

Directory of Open Access Journals (Sweden)

Maria Abildgaard Steffensen

Full Text Available Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii. To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

Chemosensitizing tumor cells by targeting the Fanconi anemia pathway with an adenovirus overexpressing dominant-negative FANCA.

Science.gov (United States)

Ferrer, Miriam; de Winter, Johan P; Mastenbroek, D C Jeroen; Curiel, David T; Gerritsen, Winald R; Giaccone, Giuseppe; Kruyt, Frank A E

2004-08-01

Fanconi anemia (FA) is a rare genetic disorder characterized by bone-marrow failure and cellular hypersensitivity to crosslinking agents, including cisplatin. Here, we studied the use of the FA pathway as a possible target for cancer gene therapy with the aim to sensitize tumor cells for cisplatin by interfering with the FA pathway. As proof-of-principle, FA and non-FA lymphoblast-derived tumors were grown subcutaneously in scid mice and treated with two different concentrations of cisplatin. As predicted, the antitumor response was considerably improved in FA tumors. An adenoviral vector encoding a dominant-negative form of FANCA, FANCA600DN, was generated that interfered with endogenous FANCA-FANCG interaction resulting in the disruption of the FA pathway as illustrated by disturbed FANCD2 monoubiquitination. A panel of cell lines, including non-small-cell lung cancer cells, could be sensitized approximately two- to three-fold for cisplatin after Ad.CMV.FANCA600DN infection that may increase upon enhanced infection efficiency. In conclusion, targeting the FA pathway may provide a novel strategy for the sensitization of solid tumors for cisplatin and, in addition, provides a tool for examining the role of the FA pathway in determining chemoresistance in different tumor types.

ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

Directory of Open Access Journals (Sweden)

Lacher Markus D

2011-07-01

CAR promoter is positively regulated through conserved ETS and CRE elements. Conclusions This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.

A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation

DEFF Research Database (Denmark)

Frandsen, Rasmus John Normand

2011-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transform......Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient......-regulation of gene expression. This review summarizes the technical advances within the field from 1998 to the summer of 2011, focusing on the development of binary vectors that are compatible with fungal transformation (over 180 general vectors) and methods for constructing binary vectors for targeted integration...

Using support vector machine ensembles for target audience classification on Twitter.

Science.gov (United States)

Lo, Siaw Ling; Chiong, Raymond; Cornforth, David

2015-01-01

The vast amount and diversity of the content shared on social media can pose a challenge for any business wanting to use it to identify potential customers. In this paper, our aim is to investigate the use of both unsupervised and supervised learning methods for target audience classification on Twitter with minimal annotation efforts. Topic domains were automatically discovered from contents shared by followers of an account owner using Twitter Latent Dirichlet Allocation (LDA). A Support Vector Machine (SVM) ensemble was then trained using contents from different account owners of the various topic domains identified by Twitter LDA. Experimental results show that the methods presented are able to successfully identify a target audience with high accuracy. In addition, we show that using a statistical inference approach such as bootstrapping in over-sampling, instead of using random sampling, to construct training datasets can achieve a better classifier in an SVM ensemble. We conclude that such an ensemble system can take advantage of data diversity, which enables real-world applications for differentiating prospective customers from the general audience, leading to business advantage in the crowded social media space.

Using support vector machine ensembles for target audience classification on Twitter.

Directory of Open Access Journals (Sweden)

Siaw Ling Lo

Full Text Available The vast amount and diversity of the content shared on social media can pose a challenge for any business wanting to use it to identify potential customers. In this paper, our aim is to investigate the use of both unsupervised and supervised learning methods for target audience classification on Twitter with minimal annotation efforts. Topic domains were automatically discovered from contents shared by followers of an account owner using Twitter Latent Dirichlet Allocation (LDA. A Support Vector Machine (SVM ensemble was then trained using contents from different account owners of the various topic domains identified by Twitter LDA. Experimental results show that the methods presented are able to successfully identify a target audience with high accuracy. In addition, we show that using a statistical inference approach such as bootstrapping in over-sampling, instead of using random sampling, to construct training datasets can achieve a better classifier in an SVM ensemble. We conclude that such an ensemble system can take advantage of data diversity, which enables real-world applications for differentiating prospective customers from the general audience, leading to business advantage in the crowded social media space.

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Science.gov (United States)

Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

1996-07-01

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

Science.gov (United States)

To, Kelvin K W; Tse, Herman; Chan, Wan-Mui; Choi, Garnet K Y; Zhang, Anna J X; Sridhar, Siddharth; Wong, Sally C Y; Chan, Jasper F W; Chan, Andy S F; Woo, Patrick C Y; Lau, Susanna K P; Lo, Janice Y C; Chan, Kwok-Hung; Cheng, Vincent C C; Yuen, Kwok-Yung

2014-12-01

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

Directory of Open Access Journals (Sweden)

Kelvin K W To

2014-12-01

Full Text Available Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1 was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention

Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica.

Science.gov (United States)

Park, Yon Mi; Kim, Jeong-Hoon; Gu, Se Hun; Lee, Sook Young; Lee, Min-Goo; Kang, Yoon Kyoo; Kang, Sung-Ho; Kim, Hak Jun; Song, Jin-Won

2012-01-05

Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica. Copyright © 2011 Elsevier Inc. All rights reserved.

Dual effects of adenovirus-mediated thrombopoietin gene transfer on hepatic oval cell proliferation and platelet counts

International Nuclear Information System (INIS)

Ichiba, Miho; Shimomura, Takashi; Murai, Rie; Hashiguchi, Koichi; Saeki, Toshiya; Yoshida, Yoko; Kanbe, Takamasa; Tanabe, Naotada; Tsuchiya, Hiroyuki; Miura, Norimasa; Tajima, Fumihito; Kurimasa, Akihiro; Hamada, Hirofumi; Shiota, Goshi

2005-01-01

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P < 0.05) and reached its plateau at 9 days (P < 0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells

Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China

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Zhu Yuan-Mao

2011-12-01

Full Text Available Abstract Background Bovine adenovirus type 3 (BAV-3 belongs to the Mastadenovirus genus of the family Adenoviridae and is involved in respiratory and enteric infections of calves. The isolation of BAV-3 has not been reported prior to this study in China. In 2009, there were many cases in cattle showing similar clinical signs to BAV-3 infection and a virus strain, showing cytopathic effect in Madin-Darby bovine kidney cells, was isolated from a bovine nasal swab collected from feedlot cattle in Heilongjiang Province, China. The isolate was confirmed as a bovine adenovirus type 3 by PCR and immunofluorescence assay, and named as HLJ0955. So far only the complete genome sequence of prototype of BAV-3 WBR-1 strain has been reported. In order to further characterize the Chinese isolate HLJ0955, the complete genome sequence of HLJ0955 was determined. Results The size of the genome of the Chinese isolate HLJ0955 is 34,132 nucleotides in length with a G+C content of 53.6%. The coding sequences for gene regions of HLJ0955 isolate were similar to the prototype of BAV-3 WBR-1 strain, with 80.0-98.6% nucleotide and 87.5-98.8% amino acid identities. The genome of HLJ0955 strain contains 16 regions and four deletions in inverted terminal repeats, E1B region and E4 region, respectively. The complete genome and DNA binding protein gene based phylogenetic analysis with other adenoviruses were performed and the results showed that HLJ0955 isolate belonged to BAV-3 and clustered within the Mastadenovirus genus of the family Adenoviridae. Conclusions This is the first study to report the isolation and molecular characterization of BAV-3 from cattle in China. The phylogenetic analysis performed in this study supported the use of the DNA binding protein gene of adenovirus as an appropriate subgenomic target for the classification of different genuses of the family Adenoviridae on the molecular basis. Meanwhile, a large-scale pathogen and serological epidemiological

Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

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Anitra L Farrow

2014-08-01

Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5 vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83. This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes

Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

OpenAIRE

Ostapchuk, Philomena; Yang, Jihong; Auffarth, Ece; Hearing, Patrick

2005-01-01

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a ro...

BS69 : A novel adenovirus E1A-associated protein that inhibits E1A transactivation

NARCIS (Netherlands)

Hateboer, G.; Gennissen, A.M.C.; Ramos, Y.F.M.; Kerkhoven, R.; Sonntag-Buck, V.; Stunnenberg, H.G.; Bernards, R.A.

1995-01-01

The adenovirus ElA gene products are nuclear phosphoproteins that can transactivate the other adenovirus early genes as well as several cellular genes, and can transform primary rodent cells in culture. Transformation and transactivation by ElA proteins is most likely to be mediated through

Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents

International Nuclear Information System (INIS)

Shashkova, Elena V.; May, Shannon M.; Barry, Michael A.

2009-01-01

Human adenovirus type 5 (Ad5) has been the most popular platform for the development of oncolytic Ads. Alternative Ad serotypes with low seroprevalence might allow for improved anticancer efficacy in Ad5-immune patients. We studied the safety and efficacy of rare serotypes Ad6, Ad11 and Ad35. In vitro cytotoxicity of the Ads correlated with expression of CAR and CD46 in most but not all cell lines. Among CAR-binding viruses, Ad5 was often more active than Ad6, among CD46-binding viruses Ad35 was generally more cytotoxic than Ad11 in cell culture studies. Ad5, Ad6, and Ad11 demonstrated similar anticancer activity in vivo, whereas Ad35 was not efficacious. Hepatotoxicity developed only in Ad5-injected mice. Predosing with Ad11 and Ad35 did not increase infection of hepatocytes with Ad5-based vector demonstrating different interaction of these Ads with Kupffer cells. Data obtained in this study suggest developing Ad6 and Ad11 as alternative Ads for anticancer treatment.

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

Science.gov (United States)

Gülen, D; Maas, S; Julius, H; Warkentin, P; Britton, H; Younos, I; Senesac, J; Pirruccello, Samuel M; Talmadge, J E

2012-05-01

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs. Copyright © 2012 Elsevier B.V. All rights reserved.

Deaths from Adenovirus in the US Military

Centers for Disease Control (CDC) Podcasts

2012-03-26

Dr. Joel Gaydos, science advisor for the Armed Forces Health Surveillance Center, and Dr. Robert Potter, a research associate for the Armed Forces Medical Examiner System, discuss deaths from adenovirus in the US military.  Created: 3/26/2012 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 3/29/2012.

Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

Directory of Open Access Journals (Sweden)

Saki Kondo

Full Text Available Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs, are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

Science.gov (United States)

Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

2000-04-01

Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

Infection with E1B-mutant adenovirus stabilizes p53 but blocks p53 acetylation and activity through E1A

DEFF Research Database (Denmark)

Savelyeva, I.; Dobbelstein, M.

2011-01-01

to the suppression of p21 transcription. Depending on the E1A conserved region 3, E1B-defective adenovirus impaired the ability of the transcription factor Sp1 to bind the p21 promoter. Moreover, the amino terminal region of E1A, binding the acetyl transferases p300 and CREB-binding protein, blocked p53 K382...... accumulation of p53, without obvious defects in p53 localization, phosphorylation, conformation and oligomerization. Nonetheless, p53 completely failed to induce its target genes in this scenario, for example, p21/CDKN1A, Mdm2 and PUMA. Two regions of the E1A gene products independently contributed...... acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p21 mRNA levels. Our findings argue that adenovirus attenuates p53-mediated p21 induction, through at least two E1B-independent mechanisms. Other virus species and cancer cells may employ analogous...

Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

Science.gov (United States)

Suzuki, Takeo; Kawamura, Kiyoko; Li, Quanhai; Okamoto, Shinya; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Yamaguchi, Naoto; Tagawa, Masatoshi

2014-09-25

Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the β-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product

Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

Energy Technology Data Exchange (ETDEWEB)

Li, Xiaohua [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Fan, Rui [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Zou, Xue [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Gao, Lin [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Jin, Haifeng [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Du, Rui [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Xia, Lin [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China); Fan, Daiming [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 17 Changle Western Road, Xi' an 710032 (China)

2007-06-29

Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.

Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

International Nuclear Information System (INIS)

Li, Xiaohua; Fan, Rui; Zou, Xue; Gao, Lin; Jin, Haifeng; Du, Rui; Xia, Lin; Fan, Daiming

2007-01-01

Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma

uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors.

Science.gov (United States)

Sobrevals, Luciano; Mato-Berciano, Ana; Urtasun, Nerea; Mazo, Adela; Fillat, Cristina

2014-01-01

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133(+) population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells. © 2013.

Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma

NARCIS (Netherlands)

Kuhlmann, K.F.D.; Geer, M.A. van; Bakker, C.T.; Dekker, J.E.M.; Havenga, M.J.E.; Oude Elferink, R.P.J.; Gouma, D.J.; Bosma, P.J.; Wesseling, J.G.

2009-01-01

Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved

cis and trans requirements for the selective packaging of adenovirus type 5 DNA.

OpenAIRE

Gräble, M; Hearing, P

1992-01-01

Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DN...

Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist Alpha-Galactosylceramide as Adjuvant

Directory of Open Access Journals (Sweden)

Shailbala Singh

2014-10-01

Full Text Available Gene-based vaccination strategies, specifically viral vectors encoding vaccine immunogens are effective at priming strong immune responses. Mucosal routes offer practical advantages for vaccination by ease of needle-free administration, and immunogen delivery at readily accessible oral/nasal sites to efficiently induce immunity at distant gut and genital tissues. However, since mucosal tissues are inherently tolerant for induction of immune responses, incorporation of adjuvants for optimal mucosal vaccination strategies is important. We report here the effectiveness of alpha-galactosylceramide (α-GalCer, a synthetic glycolipid agonist of natural killer T (NKT cells, as an adjuvant for enhancing immunogenicity of vaccine antigens delivered using viral vectors by mucosal routes in murine and nonhuman primate models. Significant improvement in adaptive immune responses in systemic and mucosal tissues was observed by including α-GalCer adjuvant for intranasal immunization of mice with vesicular stomatitis virus vector encoding the model antigen ovalbumin and adenoviral vectors expressing HIV env and Gag antigens. Activation of NKT cells in systemic and mucosal tissues along with significant increases in adaptive immune responses were observed in rhesus macaques immunized by intranasal and sublingual routes with protein or adenovirus vectored antigens when combined with α-GalCer adjuvant. These results support the utility of α-GalCer adjuvant for enhancing immunogenicity of mucosal vaccines delivered using viral vectors.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Science.gov (United States)

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Directory of Open Access Journals (Sweden)

Yanwei Bi

2014-05-01

Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

Science.gov (United States)

Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

2012-12-15

The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

Preparation and characterization of magnetic gene vectors for targeting gene delivery

Energy Technology Data Exchange (ETDEWEB)

Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

2012-10-15

Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC)

International Nuclear Information System (INIS)

Levitzki, Alexander

2012-01-01

Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody.

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

Science.gov (United States)

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

[Outbreak of follicular conjunctivitis caused by adenovirus in a geriatric centre].

Science.gov (United States)

Artieda, Juncal; Montes, Milagrosa; Vicente, Diego; Martínez, Consuelo; Piñeiro, Luis; Mendiola, Josune

2010-12-01

Adenovirus serotype 4a is a respiratory virus that occasionally causes conjunctivitis. This paper describes an outbreak of follicular conjunctivitis that occurred in a geriatric centre. Outbreak description and epidemiological research through a survey. For the microbiological study conjunctival swabs were collected using viral and bacterial transport media. Adenovirus was detected by real-time polymerase chain reaction. The serotype was determined by sequencing of a fragment of the hexon and E1 genes. In autumn 2008 an outbreak of follicular conjunctivitis caused by adenovirus serotype 4a was detected. Twenty three percent 23% (69/300) of residents and 5% (9/180) of workers in a geriatric centre in Gipuzkoa were affected. The clinical symptoms were of prolonged duration (11±5 days). The temporal association of the cases suggested transmission from person to person. The sanitary measures established (asepsis and frequent hand washing, cleaning and disinfection of objects and surfaces) were effective, interrupting the transmission of the disease within a short period of time. Rapid detection, identification of the causative agent and implementing appropriate control measures can significantly reduce the impact on both health and economic costs of these outbreaks. Copyright © 2009 Elsevier España, S.L. All rights reserved.

The Revolution in Viral Genomics as Exemplified by the Bioinformatic Analysis of Human Adenoviruses

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Sarah Torres

2010-06-01

Full Text Available Over the past 30 years, genomic and bioinformatic analysis of human adenoviruses has been achieved using a variety of DNA sequencing methods; initially with the use of restriction enzymes and more currently with the use of the GS FLX pyrosequencing technology. Following the conception of DNA sequencing in the 1970s, analysis of adenoviruses has evolved from 100 base pair mRNA fragments to entire genomes. Comparative genomics of adenoviruses made its debut in 1984 when nucleotides and amino acids of coding sequences within the hexon genes of two human adenoviruses (HAdV, HAdV–C2 and HAdV–C5, were compared and analyzed. It was determined that there were three different zones (1-393, 394-1410, 1411-2910 within the hexon gene, of which HAdV–C2 and HAdV–C5 shared zones 1 and 3 with 95% and 89.5% nucleotide identity, respectively. In 1992, HAdV-C5 became the first adenovirus genome to be fully sequenced using the Sanger method. Over the next seven years, whole genome analysis and characterization was completed using bioinformatic tools such as blastn, tblastx, ClustalV and FASTA, in order to determine key proteins in species HAdV-A through HAdV-F. The bioinformatic revolution was initiated with the introduction of a novel species, HAdV-G, that was typed and named by the use of whole genome sequencing and phylogenetics as opposed to traditional serology. HAdV bioinformatics will continue to advance as the latest sequencing technology enables scientists to add to and expand the resource databases. As a result of these advancements, how novel HAdVs are typed has changed. Bioinformatic analysis has become the revolutionary tool that has significantly accelerated the in-depth study of HAdV microevolution through comparative genomics.

Inhibition effect of B7-H1 gene-modified regulatory dendritic cells on thyroid-associated ophthalmopathy in mice

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Hua-Xin Chen

2014-10-01

Full Text Available AIM:To construct adenovirus vector expressing mice B7-H1 gene, transfect dendritic cells(DCs, and to study the therapeutic effect of modified DC on thyroid-associated ophthalmopathy(TAOin mice.METHODS: We designed and constructed B7-H1 gene adenovirus expression vector, and transfected DCs from mouse bone marrow, tested the phenotype and function of modified DCs, identificated its negative regulation to immune responses. The modified DCs were infected the sicked mice. And then the immunotherapeutic effect of modified DCs to TAO were tested. RESULTS: B7-H1 gene adenovirus vector was constructed and transfected DCs from bone marrow. The titer of the recombinant adenovirus was 1.8×109PFU/mL. B7-H1 gene modified DCs characteristics of regulatory DCs, could inhibit positive immune responses. The inhibition proceeding of TAO into mice infected modified DCs, was obviously prior to the control mice. The gene modified DCs, maybe become the new immunotherapy biological agent to thy TAO.CONCLUSION: We constructed the expression of mouse B7-H1 gene adenovirus expressed vector successfully, transfected DCs,by vector have properties of regulatory DCs, inhibiting positive immune response and the occurrence and development of thyroid eye disease. Gene modified DCs, reveal potent to the treatment of thyroid eye disease.

A Support Vector Learning-Based Particle Filter Scheme for Target Localization in Communication-Constrained Underwater Acoustic Sensor Networks.

Science.gov (United States)

Li, Xinbin; Zhang, Chenglin; Yan, Lei; Han, Song; Guan, Xinping

2017-12-21

Target localization, which aims to estimate the location of an unknown target, is one of the key issues in applications of underwater acoustic sensor networks (UASNs). However, the constrained property of an underwater environment, such as restricted communication capacity of sensor nodes and sensing noises, makes target localization a challenging problem. This paper relies on fractional sensor nodes to formulate a support vector learning-based particle filter algorithm for the localization problem in communication-constrained underwater acoustic sensor networks. A node-selection strategy is exploited to pick fractional sensor nodes with short-distance pattern to participate in the sensing process at each time frame. Subsequently, we propose a least-square support vector regression (LSSVR)-based observation function, through which an iterative regression strategy is used to deal with the distorted data caused by sensing noises, to improve the observation accuracy. At the same time, we integrate the observation to formulate the likelihood function, which effectively update the weights of particles. Thus, the particle effectiveness is enhanced to avoid "particle degeneracy" problem and improve localization accuracy. In order to validate the performance of the proposed localization algorithm, two different noise scenarios are investigated. The simulation results show that the proposed localization algorithm can efficiently improve the localization accuracy. In addition, the node-selection strategy can effectively select the subset of sensor nodes to improve the communication efficiency of the sensor network.

Adenovirus 36 and Obesity: An Overview.

Science.gov (United States)

Ponterio, Eleonora; Gnessi, Lucio

2015-07-08

There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed.

Adenovirus 36 and Obesity: An Overview

Directory of Open Access Journals (Sweden)

Eleonora Ponterio

2015-07-01

Full Text Available There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36. Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed.

Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells.

Science.gov (United States)

Long, Qifang; Yang, Ru; Lu, Weixian; Zhu, Weipei; Zhou, Jundong; Zheng, Cui; Zhou, Dongmei; Yu, Ling; Wu, Jinchang

2017-01-01

Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells. The adenovirus (Ad)-CD133-Cre expressing Cre recombinase under the control of the CD133 promoter and Ad-CMV-LoxP-Neo-LoxP-tBid expressing tBid under the control of the CMV promoter were successfully constructed using the Cre/LoxP switching system. The co-infection of Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre selectively induced tBid overexpression, which inhibited cell growth and triggered the cell apoptosis of CD133+ ovarian cancer stem cells. The Cre/LoxP system-mediated tBid overexpression activated the pro-apoptotic signaling pathway and augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. Furthermore, in xenograft experiments, co-infection with the two recombinant adenoviruses markedly suppressed tumor growth in vivo and promoted cell apoptosis in tumor tissues. Taken together, the present study provides evidence that the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system can effectively eliminate CD133+ ovarian cancer stem cells, representing a novel therapeutic strategy for the treatment of ovarian cancer.

Pandemic Influenza Virus 2009 H1N1 and Adenovirus in a High Risk Population of Young Adults: Epidemiology, Comparison of Clinical Presentations, and Coinfection

Science.gov (United States)

2014-01-08

a variety of pathogens. With the exception of the prior adenovirus vaccine era from 1980– 1996, adenoviruses have historically been the most common...administration of both live attenuated influenza and adenovirus vaccines , which could affect current trainee vaccine policies. In the meantime, concerns...change since the late 2011 reintroduction of adenovirus serotypes 4 and 7 vaccines in military trainees, or whether issues arise with concurrent

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus · Helin Li Pengbo Ning Zhi Lin Wulong Liang Kai Kang Lei He Yanming Zhang · More Details Abstract Fulltext PDF. The use of adenovirus vector-based ...

Adenovirus Infection in Children with Diarrhea Disease in ...

African Journals Online (AJOL)

ANNALS

Adenovirus Infection in Children with Diarrhea Disease in Northwestern. Nigeria. M. Aminu1, A. A. Ahmad1, J. U. Umoh2, M. C. de Beer3, M. D. Esona3, A. D. Steele3. 1Department of Microbiology, Faculty of Science, Ahmadu Bello University, Zaria Nigeria. 2Department of Veterinary Public Health and Preventive Medicine, ...

Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than serotype 5: Implications for ovarian cancer therapy

International Nuclear Information System (INIS)

Thoma, Clemens; Bachy, Veronique; Seaton, Patricia; Green, Nicola K.; Greaves, David R.; Klavinskis, Linda; Seymour, Leonard W.; Morrison, Joanne

2013-01-01

In a phase II/III clinical trial intraperitoneal (i.p.) administration of a group C adenovirus vector (Ad5) caused bowel adhesion formation, perforation and obstruction. However, we had found that i.p. group B, in contrast to group C adenoviruses, did not cause adhesions in nude BALB/c ovarian cancer models, prompting further investigation. Ex vivo, group B Ad11 caused lower inflammatory responses than Ad5 on BALB/c peritoneal macrophages. In vivo, i.p. Ad11 triggered short-term cytokine and cellular responses equal to Ad5 in both human CD46-positive and -negative mice. In contrast, in a long-term study of repeated i.p. administration, Ad11 caused no/mild, whereas Ad5 induced moderate/severe adhesions and substantial liver toxicity accompanied by elevated levels of IFNγ and VEGF and loss of i.p. macrophages, regardless of CD46 expression. It appears that, although i.p. Ad11 evokes immediate inflammation similar to Ad5, repeated administration of Ad11 is better tolerated and long-term fibrotic tissue remodelling is reduced. - Highlights: • i.p. Ad11 causes less long-term intraperitoneal inflammation than Ad5 in CD46-transgenic mice. • Ex vivo BALB/c peritoneal macrophages express less RANTES after Ad11 than Ad3 or Ad5 treatment. • In vivo, cytokine and cellular responses 6 h after i.p. Ad11 are equal to Ad5. • In contrast, after repeated i.p. application, Ad5, but not Ad11, causes severe i.p. toxicity. • The use of Ad11 instead of Ad5 might increase patient safety in future virotherapy of ovarian cancer

Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than serotype 5: Implications for ovarian cancer therapy

Energy Technology Data Exchange (ETDEWEB)

Thoma, Clemens, E-mail: c.thoma@oxfordalumni.org [Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU (United Kingdom); Bachy, Veronique [Peter Gorer Department of Immunobiology, Kings College London, Guys Hospital, Great Maze Pond, London SE1 9RT (United Kingdom); Seaton, Patricia [Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU (United Kingdom); Green, Nicola K. [Clinical Biomanufacturing Facility, University of Oxford, Old Road, Oxford OX3 7JT (United Kingdom); Greaves, David R. [Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Klavinskis, Linda [Peter Gorer Department of Immunobiology, Kings College London, Guys Hospital, Great Maze Pond, London SE1 9RT (United Kingdom); Seymour, Leonard W. [Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ (United Kingdom); Morrison, Joanne [Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU (United Kingdom); Department of Obstetrics and Gynaecology, Musgrove Park Hospital, Taunton TA1 5DA (United Kingdom)

2013-12-15

In a phase II/III clinical trial intraperitoneal (i.p.) administration of a group C adenovirus vector (Ad5) caused bowel adhesion formation, perforation and obstruction. However, we had found that i.p. group B, in contrast to group C adenoviruses, did not cause adhesions in nude BALB/c ovarian cancer models, prompting further investigation. Ex vivo, group B Ad11 caused lower inflammatory responses than Ad5 on BALB/c peritoneal macrophages. In vivo, i.p. Ad11 triggered short-term cytokine and cellular responses equal to Ad5 in both human CD46-positive and -negative mice. In contrast, in a long-term study of repeated i.p. administration, Ad11 caused no/mild, whereas Ad5 induced moderate/severe adhesions and substantial liver toxicity accompanied by elevated levels of IFNγ and VEGF and loss of i.p. macrophages, regardless of CD46 expression. It appears that, although i.p. Ad11 evokes immediate inflammation similar to Ad5, repeated administration of Ad11 is better tolerated and long-term fibrotic tissue remodelling is reduced. - Highlights: • i.p. Ad11 causes less long-term intraperitoneal inflammation than Ad5 in CD46-transgenic mice. • Ex vivo BALB/c peritoneal macrophages express less RANTES after Ad11 than Ad3 or Ad5 treatment. • In vivo, cytokine and cellular responses 6 h after i.p. Ad11 are equal to Ad5. • In contrast, after repeated i.p. application, Ad5, but not Ad11, causes severe i.p. toxicity. • The use of Ad11 instead of Ad5 might increase patient safety in future virotherapy of ovarian cancer.

Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor.

Science.gov (United States)

Boonthum, Chatwalee; Namdee, Katawut; Boonrungsiman, Suwimon; Chatdarong, Kaywalee; Saengkrit, Nattika; Sajomsang, Warayuth; Ponglowhapan, Suppawiwat; Yata, Teerapong

2017-02-10

The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy ( 1 H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lentiviral Delivery of a Vesicular Glutamate Transporter 1 (VGLUT1)-Targeting Short Hairpin RNA Vector Into the Mouse Hippocampus Impairs Cognition

NARCIS (Netherlands)

King, Madeleine V.; Kurian, Nisha; Qin, Si; Papadopoulou, Nektaria; Westerink, Ben H. C.; Cremers, Thomas I.; Epping-Jordan, Mark P.; Le Poul, Emmanuel; Ray, David E.; Fone, Kevin C. F.; Kendall, David A.; Marsden, Charles A.; Sharp, Tyson V.

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target

The presence of enterovirus, adenovirus, and parvovirus B19 in myocardial tissue samples from autopsies

DEFF Research Database (Denmark)

Nielsen, Trine Skov; Hansen, Jakob; Nielsen, Lars Peter

2014-01-01

of adenovirus, enterovirus, and parvovirus B19 (PVB) in myocardial autopsy samples from myocarditis related deaths and in non-inflamed control hearts in an effort to clarify their significance as the causes of myocarditis in a forensic material. METHODS: We collected all autopsy cases diagnosed with myocarditis...... from 1992 to 2010. Eighty-four suicidal deaths with morphologically normal hearts served as controls. Polymerase chain reaction was used for the detection of the viral genomes (adenovirus, enterovirus, and PVB) in myocardial tissue specimens. The distinction between acute and persistent PVB infection...... was made by the serological determination of PVB-specific immunoglobulins M and G. RESULTS: PVB was detected in 33 of 112 (29 %) myocarditis cases and 37 of 84 (44 %) control cases. All of the samples were negative for the presence of adenovirus and enterovirus. Serological evidence of an acute PVB...

SU-E-J-115: Correlation of Displacement Vector Fields Calculated by Deformable Image Registration Algorithms with Motion Parameters of CT Images with Well-Defined Targets and Controlled-Motion

Energy Technology Data Exchange (ETDEWEB)

Jaskowiak, J; Ahmad, S; Ali, I [University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Alsbou, N [Ohio Northern University, Ada, OH (United States)

2015-06-15

Purpose: To investigate correlation of displacement vector fields (DVF) calculated by deformable image registration algorithms with motion parameters in helical axial and cone-beam CT images with motion artifacts. Methods: A mobile thorax phantom with well-known targets with different sizes that were made from water-equivalent material and inserted in foam to simulate lung lesions. The thorax phantom was imaged with helical, axial and cone-beam CT. The phantom was moved with a cyclic motion with different motion amplitudes and frequencies along the superior-inferior direction. Different deformable image registration algorithms including demons, fast demons, Horn-Shunck and iterative-optical-flow from the DIRART software were used to deform CT images for the phantom with different motion patterns. The CT images of the mobile phantom were deformed to CT images of the stationary phantom. Results: The values of displacement vectors calculated by deformable image registration algorithm correlated strongly with motion amplitude where large displacement vectors were calculated for CT images with large motion amplitudes. For example, the maximal displacement vectors were nearly equal to the motion amplitudes (5mm, 10mm or 20mm) at interfaces between the mobile targets lung tissue, while the minimal displacement vectors were nearly equal to negative the motion amplitudes. The maximal and minimal displacement vectors matched with edges of the blurred targets along the Z-axis (motion-direction), while DVF’s were small in the other directions. This indicates that the blurred edges by phantom motion were shifted largely to match with the actual target edge. These shifts were nearly equal to the motion amplitude. Conclusions: The DVF from deformable-image registration algorithms correlated well with motion amplitude of well-defined mobile targets. This can be used to extract motion parameters such as amplitude. However, as motion amplitudes increased, image artifacts increased

Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

NARCIS (Netherlands)

Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

2003-01-01

Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays

OpenAIRE

Zaric, Marija; Becker, Pablo Daniel; Hervouet, Catherine; Kalcheva, Petya; Ibarzo Yus, Barbara; Cocita, Clement; O'Neill, Lauren Alexandra; Kwon, Sung-Yun; Klavinskis, Linda Sylvia

2017-01-01

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag...

Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

Directory of Open Access Journals (Sweden)

Ashish V Chintakuntlawar

2010-04-01

Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

Assessment of the Incidence of Enteric Adenovirus Species and Serotypes in Surface Waters in the Eastern Cape Province of South Africa: Tyume River as a Case Study

Directory of Open Access Journals (Sweden)

Timothy Sibanda

2012-01-01

Full Text Available TaqMan real-time PCR was used for the detection and quantitation of adenoviruses in Tyume River water samples over a 12-month period. A total of 72 samples were analysed, and 22 samples were positive for adenovirus. Of the positive samples, 18 were collected from downstream sampling points. Among the downstream sampling points, adenovirus detection rate increased with distance downstream, being 28%, 33%, and 39% for Alice, Drayini, and Manqulweni, respectively. The Alice sampling site had the highest concentrations of adenovirus ranging between 6.54×103 genome copies/L and 8.49×104 genome copies/L. The observed trend could have been expected considering the level of anthropogenic activities in areas along the lower stretch of Tyume River, with the major one being the effluent of treated and semi treated sewage from wastewater treatment facilities. Adenovirus detection was sporadic at most sampling sites. Multiplex conventional PCR was used for the detection of clinically important adenovirus species B, C, and F and their serotypes. Species C and F adenoviruses were detected in 77% and 18% of the samples, respectively. Most adenovirus positive samples were obtained from areas of increased population densities. The presence of adenoviruses may confirm the risk of its transmission to the human population.

Prevalence of rotavirus, adenovirus and astrovirus infection in young ...

African Journals Online (AJOL)

Objective: To determine the prevalence of three enteric viruses, namely rotavirus, adenovirus and astrovirus, as agents of diarrhoea in and around Gaborone, Botswana. Design: The sample were categorised into four groups according to the age of the patient: 0-3 months, 4-6 months, 7-12 months and 25-60 months.

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

Science.gov (United States)

Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

2009-11-01

Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

[Effect of topical application of a recombinant adenovirus carrying promyelocytic leukemia gene in a psoriasis-like mouse model].

Science.gov (United States)

Wang, Qiongyu; Zhang, Aijun; Ma, Huiqun; Wang, Shijie; Ma, Yunyun; Zou, Xingwei; Li, Ruilian

2013-03-01

To investigate the effects of topical treatment with adenovirus-mediated promyelocytic leukemia gene (PML) gene in a psoriasis-like mouse model. The effect of adenovirus-mediated PML gene on the granular layer of mouse tail scale epidermis and epithelial mitosis were observed on longitudinal histological sections prepared from the tail skin and vaginal epithelium of the mice. Adenovirus-mediated PML gene significantly inhibited mitosis of mouse vaginal epithelial cells and promoted the formation of granular layer in mouse tail scale epidermis. The therapeutic effect of PML gene in the psoriasis-like mouse model may be associated with increased granular cells and suppressed epidemic cell proliferation.

E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

Science.gov (United States)

Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

1999-05-01

Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

Enfermedad neurologica por adenovirus Neurologic disease due to adenovirus infection

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Cristina L. Lema

2005-06-01

Full Text Available El objetivo de este trabajo fue determinar la prevalencia de adenovirus (ADV en las infecciones del sistema nervioso central (SNC. Se analizaron 108 muestras de líquido cefalorraquídeo (LCR provenientes de 79 casos de encefalitis, 7 meningitis y 22 de otras patologías neurológicas, recibidas en el período 2000-2002. Cuarenta y nueve (47.35% se obtuvieron de pacientes inmunocomprometidos. La presencia de ADV se investigó mediante reacción en cadena de la polimerasa en formato anidado (Nested-PCR. La identificación del genogrupo se realizó mediante análisis filogenético de la secuencia nucleotídica parcial de la región que codifica para la proteína del hexón. Se detectó la presencia de ADV en 6 de 108 (5.5% muestras de LCR analizadas. Todos los casos positivos pertenecieron a pacientes con encefalitis que fueron 79, (6/79, 7.6%. No se observó diferencia estadísticamente significativa entre los casos de infección por ADV en pacientes inmunocomprometidos e inmunocompetentes (p>0.05. Las cepas de ADV detectadas se agruparon en los genogrupos B1 y C. En conclusión, nuestros resultados describen el rol de los ADV en las infecciones neurológicas en Argentina. La información presentada contribuye al conocimiento de su epidemiología, en particular en casos de encefalitis.The aim of this study was to assess the prevalence of adenovirusm (ADV infections in neurological disorders. A total of 108 cerebrospinal fluid (CSF samples from 79 encephalitis cases, 7 meningitis and 22 other neurological diseases analysed in our laboratory between 2000 and 2002 were studied. Forty nine (47.4% belonged to immunocompromised patients. Viral genome was detected using nested polymerase chain reaction (Nested-PCR and ADV genotypes were identified using partial gene sequence analysis of hexon gene. Adenovirus were detected in 6 of 108 (5.5% CSF samples tested. All of these were from encephalitis cases, 6/79, representing 7.6% of them. No statistically

Intratracheal injection of adenovirus containing the human MNSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis

International Nuclear Information System (INIS)

Epperly, Michael W.; Bray, Jenifer A.; Krager, Stephen; Berry, Luann M.; Gooding, William; Engelhardt, John F.; Zwacka, Ralf; Travis, Elizabeth L.; Greenberger, Joel S.

1999-01-01

Purpose: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. Methods and Materials: Athymic Nude (Nu/J) mice were intratracheally injected with 10 9 plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-α, TGF-β, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H and E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. Results: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry

Enhanced reactivation and mutagenesis of UV-irradiated adenovirus in normal human fibroblasts

International Nuclear Information System (INIS)

Bennett, C.B.; Rainbow, A.J.

1988-01-01

UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) for two adenovirus temperature-sensitive mutants were examined following the infection of normal human fibroblasts. UV-irradiation of the virus alone resulted in dose-dependent increase in the UV-induced reversion frequency (RF) of viral progeny and a dose-dependent exponential decrease in progeny survival, when infecting non-irradiated cells. Analysis of the slopes of the UV-induced reversion curves suggested that 2.5 ± 0.3 and 2.4 ± 0.5 'hits' were required to produce a targeted reversion event among the viral progeny of Ad5ts36 and Ad5ts125 respectively. UV-irradiation of cells 24 h prior to infection resulted in a significant increase in progeny survival for UV-irradiated virus (UVER factor = 3.4 ± 0.8) concomitant with a significant increase in RF for UV-irradiated virus (targeted increase = 1.9 ± 0.3). The UV-induced RF per lethal hit to the virus was also significantly greater in UV-irradiated compared with non-irradiated cells. These results are consistent with the existence of a UV-inducible error-prone DNA repair mechanism in normal human cells. (author)

Targeting the immune system to fight cancer using chemical receptor homing vectors carrying Poly Inosine/Cytosine (PolyIC

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Alexander eLevitzki

2012-02-01

Full Text Available Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, poly Inosine/Cytosine (polyIC, into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA binding proteins, such as dsRNA dependent protein kinase (PKR, Toll-3 receptor (TLR3, retinoic acid–inducible gene I (RIG-1 and melanoma differentiation–associated gene 5 (MDA5. The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1 Recruitment of the immune system is localized to the tumor. (2 The response is rapid, leading to fast tumor eradication. (3 The bystander effects lead to the eradication of tumor cells not harboring the target. (4 The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which be a small molecule, a single chain antibody or an

Recent Advances in Non-viral Vectors for Gene Delivery

Science.gov (United States)

Guo, Xia; Huang, Leaf

2011-01-01

CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel

Optimization and evaluation of a method to detect adenoviruses in river water

Data.gov (United States)

U.S. Environmental Protection Agency — This dataset includes the recoveries of spiked adenovirus through various stages of experimental optimization procedures. This dataset is associated with the...

A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

Directory of Open Access Journals (Sweden)

Michael C Keefer

Full Text Available We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35 vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN and env (Ad35-ENV, both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults.Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively within one of four dosage groups: Ad35-GRIN/ENV 2×10(9 (A, 2×10(10 (B, 2×10(11 (C, or Ad35-GRIN 1×10(10 (D viral particles.No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC per 10(6 PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination.Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional

The urethral smear as a tool in diagnosing adenovirus-induced urethritis.

Science.gov (United States)

Tønsberg, E; Hartgill, U

2014-12-01

Adenovirus is a recognised cause of non-gonococcal urethritis, and is not uncommonly associated with extragenital signs and symptoms. This case report describes a patient with symptoms of conjunctivitis, meatitis and urethritis. The urethral smear revealed almost exclusively monocytes microscopically, raising the suspicion of a viral aetiology. Results confirmed the presence of adenovirus in both the eyes and urethra. Despite waning reliance on the urethral smear in sexual health clinics, it can still be an important diagnostic tool in assessing the aetiology of non-specific urethritis. Finding an obvious monocytic cell response in the urethral smear can indicate a viral cause and allow the clinician to optimise management, counsel appropriately, and potentially reduce unnecessary antibiotic use. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Sequence typing of adenovirus from samples from hematological stem cell transplant recipients.

Science.gov (United States)

Al Qurashi, Yasir Mohammed A; Guiver, Malcolm; Cooper, Robert J

2011-11-01

Adenovirus infections are usually mild or even asymptomatic, but infections with the virus are being recognized increasingly as a major cause of mortality and morbidity in the immunocompromised, particularly hematological stem cell transplant patients where infections can be life threatening and mortality may reach 60%. Typing by sequencing the HVR7 region of the hexon was established and validated using 60 isolates of different serotypes from the six of the seven species which had been typed previously by serum neutralization. Analysis of nucleotide sequences was used to type 227 samples from 41 hematological stem cell transplant recipients. Types from six species were detected but species C types were detected in 51.4% and species A in 34.3% of patients. Seven patients were infected with different adenovirus types sequentially and a further six patients had evidence of simultaneous multiple infections. Many of the sequences had several differences from the prototype strains which will allow tracing of outbreaks and provide evidence for cross-infection in a hospital setting. In this study, the phylogenetic analysis of adenovirus sequences from hematological stem cell transplant patients' samples showed evidence of two possible cross-infection incidents involving three and five patients, respectively. Copyright © 2011 Wiley-Liss, Inc.

Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

Energy Technology Data Exchange (ETDEWEB)

Guardado Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Langlois, Patrick [Agence Francaise de Securité Sanitaire des Aliments, Unité Génétique Virale et Biosecurité, Site Les Croix, BP 53, F-22440 Ploufragan (France); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

2006-05-01

Avian adenovirus long-fibre head trimers were expressed, purified and crystallized. The crystals belong to space group C2 (unit-cell parameters a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°). A complete highly redundant data set was collected to 2.2 Å resolution at 100 K using a rotating-anode X-ray source. Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579–793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10 000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kα radiation to at least 1.9 Å resolution and a complete data set was collected from a single crystal to 2.2 Å resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress.

How effective is integrated vector management against malaria and lymphatic filariasis where the diseases are transmitted by the same vector?

OpenAIRE

Stone, C.; Lindsay, S.W.; Chitnis, N.

2014-01-01

Background: The opportunity to integrate vector management across multiple vector-borne diseases is particularly plausible for malaria and lymphatic filariasis (LF) control where both diseases are transmitted by the same vector. To date most examples of integrated control targeting these diseases have been unanticipated consequences of malaria vector control, rather than planned strategies that aim to maximize the efficacy and take the complex ecological and biological interactions between th...

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

Science.gov (United States)

Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D; Chorro, Laurent; Carlin, Leo M; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S

2013-02-19

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management.

Science.gov (United States)

Pocquet, Nicolas; Darriet, Frédéric; Zumbo, Betty; Milesi, Pascal; Thiria, Julien; Bernard, Vincent; Toty, Céline; Labbé, Pierrick; Chandre, Fabrice

2014-07-01

Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. The low resistance observed in Mayotte's main disease vectors is particularly interesting, because it leaves a range of tools useable by vector control

Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

Science.gov (United States)

Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

2012-02-01

Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. Copyright © 2011 Elsevier Inc. All rights reserved.

Measurement of the Electric Form Factor of the Neutron through d-vector(e-vector, e'n)p

International Nuclear Information System (INIS)

Hongguo Zhu; Abdellah Ahmidouch; Anklin, H.; Arenhoevel, H.; Chris Armstrong; C. Bernet; Werner Boeglin; Herbert Breuer; Paul Brindza; D. Brown; S. Bueltmann; Roger Carlini; Nicholas Chant; Anthony Cowley; Donald Crabb; Samuel Danagoulian; D. B. Day; Tom Eden; Rolf Ent; Yusuf Farah; Renee Fatimi; Kenneth Garrow; Chris Harris; Markus Hauger; A. Honegger; J. Jourdan; M. Kaufmann; Mahbub Khandaker; Gunther Kubon; Jechiel Lichtenstadt; Richard Lindgren; Robert Lourie; Allison Lung; David Mack; Sudhir Malik; Pete Markowitz; Kenneth McFarlane; Paul McKee; Dustin McNulty; Geoffrey Milanovich; Joseph Mitchell; Hamlet Mkrtchyan; M. Muehlbauer; Thomas Petitjean; Yelena Prok; Daniela Rohe; Emmanuel Rollinde; Oscar Rondon-Aramayo; Philip Roos; Reyad Sawafta; Ingo Sick; C. Smith; Tim Southern; Michael Steinacher; Stepan Stepanyan; Vardan Tadevosyan; Raphael Tieulent; Al Tobias; Bill Vulcan; Glen Warren; H. Woehrle; Stephen Wood; Chen Yan; Marko Zeier; Jianguo Zhao; Beni Zihlmann

2001-01-01

We report the first measurement using a solid polarized target of the neutron electric form factor G n E via d-vector(e-vector, e'n)p. G n E was determined from the beam-target asymmetry in the scattering of longitudinally polarized electrons from polarized deuterated ammonia ( 15 ND 3 ). The measurement was performed in Hall C at Thomas Jefferson National Accelerator Facility in quasifree kinematics with the target polarization perpendicular to the momentum transfer. The electrons were detected in a magnetic spectrometer in coincidence with neutrons in a large solid angle segmented detector. We find G n E = 0.04632±0.00616(stat)±0.00341(syst) at Q 2 = 0.495 (GeV/c) 2

Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Science.gov (United States)

Niu, Qingli; Hou, Wei; Churchyard, Gavin; Nitayaphan, Sorachai; Pitisuthithum, Punnee; Rerks-Ngarm, Supachai; Franchini, Genoveffa

2018-01-01

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination. PMID:29474461

morphological identification of malaria vectors within anopheles

African Journals Online (AJOL)

DR. AMIN

Africa among the human population. Determination of risk of malaria transmission requires quick and accurate methods of identification of Anopheles mosquitoes especially when targeting vector control. (Maxwell, et al., 2003). Anopheles mosquito transmits malaria. The most important vectors of malaria are members of.

Characterization and vectorization of siRNA targeting RET/PTC1 in human papillary thyroid carcinoma cells

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Massade L.

2011-10-01

Full Text Available RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC and represents a good target for small interfering RNA (siRNA. Our aim was: i to target the RET/PTC1 oncogene by siRNAs, ii to assess the knockdown effects on cell growth and cell cycle regulation and iii to vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.

Effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mDEF201 on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.

Directory of Open Access Journals (Sweden)

Donald F Smee

Full Text Available An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201 was evaluated for efficacy against lethal cowpox (Brighton strain and vaccinia (WR strain virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl and pulmonary (50-µl, to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI, upper respiratory infections (URI, and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term or 56d (long-term prior to virus challenge. Single nasal sinus treatments of 10(6 and 10(7 PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201. Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7 mDEF201 treatments, but a 10(6 dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7 PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5 to 10(7 PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

Molecular epidemiology of human adenovirus infections in Denmark, 2011–2016

DEFF Research Database (Denmark)

Barnadas, Céline; Schmidt, Dennis Jelsbak; Fischer, Thea K.

2018-01-01

Background: Human adenoviruses (HAdVs) can cause respiratory tract infections, conjunctivitis, diarrhoea and outbreaks have been reported. However, little is known about the disease burden and the molecular epidemiology of HAdV. Objectives: To retrospectively perform a molecular characterization ...

Characteristics of adenovirus urethritis among heterosexual men and men who have sex with men: a review of clinical cases.

Science.gov (United States)

Samaraweera, Geethani R; Garcia, Katherine; Druce, Julian; Williams, Henrietta; Bradshaw, Catriona S; Fairley, Christopher K; Chow, Eric Pf; Denham, Ian M; Read, Timothy R H; Chen, Marcus Y

2016-05-01

The aim of this study was to characterise the clinical features of adenovirus urethritis in men and to compare the frequency of these between heterosexual men and men who have sex with men (MSM). This was a review of the clinical and laboratory information from men diagnosed with PCR-confirmed adenovirus urethritis at the Melbourne Sexual Health Centre between January 2006 and April 2014. 102 adenovirus urethritis cases were reported, among which 61 were heterosexual men and 41 MSM. Eighty-nine per cent (n=91) had signs of meatitis or conjunctivitis: 51% had meatitis only; 32% meatitis together with conjunctivitis and 6% with conjunctivitis only. The distribution of symptoms and signs was similar among heterosexual men and MSM (p values >0.1). Adenovirus was the sole pathogen found in 93% of cases, excluding gonorrhoea, chlamydia, Mycoplasma genitalium and herpes simplex virus. Only 37% had ≥5 polymorphs per high-power field from a urethral smear. Where samples were still available for adenoviral sequencing (n=20), all were subgroup D. The clinical features of adenovirus urethritis in men can be distinctive and aid diagnosis, distinguishing it from other treatable causes of male urethritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Formation of a Multiple Protein Complex on the Adenovirus Packaging Sequence by the IVa2 Protein▿

OpenAIRE

Tyler, Ryan E.; Ewing, Sean G.; Imperiale, Michael J.

2007-01-01

During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first ...

AAV Vectorization of DSB-mediated Gene Editing Technologies.

Science.gov (United States)

Moser, Rachel J; Hirsch, Matthew L

2016-01-01

Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.