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Sample records for adenovirus vector transfer

  1. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

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    Julius W Kim

    Full Text Available Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  2. 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

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

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    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

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

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    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.

  5. Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors.

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    Abbink, Peter; Maxfield, Lori F; Ng'ang'a, David; Borducchi, Erica N; Iampietro, M Justin; Bricault, Christine A; Teigler, Jeffrey E; Blackmore, Stephen; Parenteau, Lily; Wagh, Kshitij; Handley, Scott A; Zhao, Guoyan; Virgin, Herbert W; Korber, Bette; Barouch, Dan H

    2015-02-01

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors. Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Chimpanzee Adenovirus Vector Ebola Vaccine.

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    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

  7. An Update on Canine Adenovirus Type 2 and Its Vectors

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    Bru, Thierry; Salinas, Sara; Kremer, Eric J.

    2010-01-01

    Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors. PMID:21994722

  8. Adenovirus-vectored Ebola vaccines.

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    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.

  9. Getting genetic access to natural adenovirus genomes to explore vector diversity.

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    Zhang, Wenli; Ehrhardt, Anja

    2017-10-01

    Recombinant vectors based on the human adenovirus type 5 (HAdV5) have been developed and extensively used in preclinical and clinical studies for over 30 years. However, certain restrictions of HAdV5-based vectors have limited their clinical applications because they are rather inefficient in specifically transducing cells of therapeutic interest that lack the coxsackievirus and adenovirus receptor (CAR). Moreover, enhanced vector-associated toxicity and widespread preexisting immunity have been shown to significantly hamper the effectiveness of HAdV-5-mediated gene transfer. However, evolution of adenoviruses in the natural host is driving the generation of novel types with altered virulence, enhanced transmission, and altered tissue tropism. As a consequence, an increasing number of alternative adenovirus types were identified, which may represent a valuable resource for the development of novel vector types. Thus, researchers are focusing on the other naturally occurring adenovirus types, which are structurally similar but functionally different from HAdV5. To this end, several strategies have been devised for getting genetic access to adenovirus genomes, resulting in a new panel of adenoviral vectors. Importantly, these vectors were shown to have a host range different from HAdV5 and to escape the anti-HAdV5 immune response, thus underlining the great potential of this approach. In summary, this review provides a state-of-the-art overview of one essential step in adenoviral vector development.

  10. 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.

  11. 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.

  12. 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.

  13. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Improvement of oncolytic adenovirus vectors through genetic capsid modifications

    NARCIS (Netherlands)

    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

  16. Progress on adenovirus-vectored universal influenza vaccines.

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    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 (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

  17. Alternate adenovirus type-pairs for a possible circumvention of host immune response to recombinant adenovirus vectors.

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    Nász, I; Adám, E; Lengyel, A

    2001-01-01

    With the help of monoclonal antibodies the existence of at least 18 different earlier not known intertype (IT) specific epitopes were demonstrated in different numbers and combinations on the hexons of different adenovirus serotypes. The IT specific epitopes play an important role in the experimental gene therapy and in the recombinant adenovirus vaccination because of the harmful immune response of the recipient organisms directed against the many different epitopes of the adenovirus vector. For the elimination of harmful effect the authors suggest the use of multiple vectors, each prepared from different adenovirus serotypes showing the loosest antigenic relationship to each other. The vectors would be used sequentially when second or multiple administration is needed. For this purpose the authors determined and described 31 such adenovirus type-pairs, which are probably the best alternates for sequential use in experimental gene therapy.

  18. A novel technology to target adenovirus vectors : application in cells involved in atherosclerosis

    NARCIS (Netherlands)

    Gras, Jan Cornelis Emile

    2007-01-01

    In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR

  19. 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...

  20. 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.

  1. Non-Replicating Adenovirus-Vectored Anthrax Vaccine

    International Nuclear Information System (INIS)

    Van Kampen, K. R.; Zhang, J.; Jex, E.; Tang, D. C.

    2007-01-01

    As bioterrorism is emerging as a national threat, it is urgent to develop a new generation of anthrax vaccines that can be rapidly produced and mass administered in an emergency setting. We have demonstrated that protective immunity against anthrax spores could be elicited in mice by intranasal administration of a non-replicating human adenovirus serotype 5 (Ad5)-derived vector encoding Bacillus anthracis protective antigen (PA) in a single-dose regimen. The potency of an Ad5 vector encoding PA was remarkably enhanced by codon optimization of the PA gene to match the tRNA pool found in human cells. This nasal vaccine can be mass-administered by non-medical personnel during a bioterrorist attack. In addition, replication-competent adenovirus (RCA)-free Ad5-vectored anthrax vaccines can be mass produced in PER.C6 cells in serum-free wave bioreactors and purified by column chromatography to meet a surge in demand. The non-replicating nature of this new generation of anthrax vaccine ensures an excellent safety profile for vaccines and the environment.(author)

  2. Recent advances in genetic modification of adenovirus vectors for cancer treatment.

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    Yamamoto, Yuki; Nagasato, Masaki; Yoshida, Teruhiko; Aoki, Kazunori

    2017-05-01

    Adenoviruses are widely used to deliver genes to a variety of cell types and have been used in a number of clinical trials for gene therapy and oncolytic virotherapy. However, several concerns must be addressed for the clinical use of adenovirus vectors. Selective delivery of a therapeutic gene by adenovirus vectors to target cancer is precluded by the widespread distribution of the primary cellular receptors. The systemic administration of adenoviruses results in hepatic tropism independent of the primary receptors. Adenoviruses induce strong innate and acquired immunity in vivo. Furthermore, several modifications to these vectors are necessary to enhance their oncolytic activity and ensure patient safety. As such, the adenovirus genome has been engineered to overcome these problems. The first part of the present review outlines recent progress in the genetic modification of adenovirus vectors for cancer treatment. In addition, several groups have recently developed cancer-targeting adenovirus vectors by using libraries that display random peptides on a fiber knob. Pancreatic cancer-targeting sequences have been isolated, and these oncolytic vectors have been shown by our group to be associated with a higher gene transduction efficiency and more potent oncolytic activity in cell lines, murine models, and surgical specimens of pancreatic cancer. In the second part of this review, we explain that combining cancer-targeting strategies can be a promising approach to increase the clinical usefulness of oncolytic adenovirus vectors. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials.

    Science.gov (United States)

    Kallel, Héla; Kamen, Amine A

    2015-05-01

    Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines. This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing. Additionally, advancements in vector characterization will greatly facilitate the development of novel vectored vaccine candidates. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  11. 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

  12. 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...

  13. 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

  14. Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Johanna P Laakkonen

    Full Text Available In central nervous system (CNS-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad vectors across the blood-brain barrier (BBB in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii the attachment site of the ligand influences its transcytosis efficiency and iii combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.

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

    Science.gov (United States)

    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...

  16. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    Directory of Open Access Journals (Sweden)

    E. Quattrocchi

    2011-09-01

    Full Text Available Collagen Induced Arthritis (CIA is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig, which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or β-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2×108 pfu suppressed established CIA, whereas the control β-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNg production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

  17. Covalent decoration of adenovirus vector capsids with the carbohydrate epitope αGal does not improve vector immunogenicity, but allows to study the in vivo fate of adenovirus immunocomplexes.

    Science.gov (United States)

    Kratzer, Ramona F; Espenlaub, Sigrid; Hoffmeister, Andrea; Kron, Matthias W; Kreppel, Florian

    2017-01-01

    Adenovirus-based vectors are promising tools for genetic vaccination. However, several obstacles have to be overcome prior to a routine clinical application of adenovirus-based vectors as efficacious vectored vaccines. The linear trisaccharide epitope αGal (alpha-Gal) with the carbohydrate sequence galactose-α-1,3-galactosyl-β-1,4-N-acetylglucosamine has been described as a potent adjuvant for recombinant or attenuated vaccines. Humans and α-1,3-galactosyltransferase knockout mice do not express this epitope. Upon exposure of α-1,3-galactosyltransferase-deficient organisms to αGal in the environment, large amounts of circulating anti-Gal antibodies are produced consistently. Immunocomplexes formed between recombinant αGal-decorated vaccines and anti-Gal antibodies exhibit superior immunogenicity. We studied the effects of the trisaccharide epitope on CD8 T cell responses that are directed specifically to vector-encoded transgenic antigens. For that, covalently αGal-decorated adenovirus vectors were delivered to anti-Gal α-1,3-galactosyltransferase knockout mice. We generated replication-defective, E1-deleted adenovirus type 5 vectors that were decorated with αGal at the hexon hypervariable regions 1 or 5, at fiber knob, or at penton base. Surprisingly, none of the adenovirus immunocomplexes being formed from αGal-decorated adenovirus vectors and anti-Gal immunoglobulins improved the frequencies of CD8 T cell responses against the transgenic antigen ovalbumin. Humoral immunity directed to the adenovirus vector was neither increased. However, our data indicated that decoration of Ad vectors with the αGal epitope is a powerful tool to analyze the fate of adenovirus immunocomplexes in vivo.

  18. 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...

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

    Science.gov (United States)

    Warimwe, George M; Lorenzo, Gema; Lopez-Gil, Elena; Reyes-Sandoval, Arturo; Cottingham, Matthew G; Spencer, Alexandra J; Collins, Katharine A; Dicks, Matthew D J; Milicic, Anita; Lall, Amar; Furze, Julie; Turner, Alison V; Hill, Adrian V S; Brun, Alejandro; Gilbert, Sarah C

    2013-12-05

    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 antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

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

    Science.gov (United States)

    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.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Gene therapy in the inner ear using adenovirus vectors.

    Science.gov (United States)

    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.

  4. 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.

  5. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    Directory of Open Access Journals (Sweden)

    Briana Jill Williams

    Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

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

    Science.gov (United States)

    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.

  7. 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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome

    Directory of Open Access Journals (Sweden)

    Kahori Shimizu

    2014-01-01

    Full Text Available Leaky expression of adenovirus (Ad genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3′-untranslated region (UTR of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a–targeted sequences into the 3′-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a–mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

  12. 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

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

    Science.gov (United States)

    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.

  14. 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

  15. 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...

  16. Uptake of 131I-FIAU in BMSCs infected by adenovirus vector-mediated HSV1-TK

    International Nuclear Information System (INIS)

    Zhang Binqing; Wu Tao; Sun Xun; An Rui

    2010-01-01

    Report gene HSV1-TK and therapy gene were connected by IRES, and recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP was constructed and infected with BMSCs at MOI of 0, 50, 100, 150, 200 and 250, with the control recombinant adenovirus vector of Ad5-EGFP. Green fluorescence cell positive rate was observed under the microscopy. MTT assay was used to determine the cell proliferation. bFGF and EGF were used to induce the BMSCs, and RQ-PCR to determine target gene expression in infection BMSCs. Uptake of 131 I-FIAU was assessed by gamma counter. The data were processed by SPSS11.code. Recombinant adenovirus at MOI 150 had high infectionefficiency and low toxic in BMSCs. There was a strong relation between the mRNA expression of TK and BDNF in infection BMSCs. The significance between the infection BMSCs and control BMSCs for uptake of 131 I-FIAU at all the time points was t=23.06-173.83 and P 131 I-FIAU. This suggests a suitable gene vector for tracing genetically modified stem cells. (authors)

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

    Science.gov (United States)

    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.

  18. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. 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.

  5. 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

    2014-08-21

    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.

  6. 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

  7. Lentiviral Vector Gene Transfer to Porcine Airways

    Directory of Open Access Journals (Sweden)

    Patrick L Sinn

    2012-01-01

    Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

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

    Science.gov (United States)

    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.

  9. 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

  10. Radioiodine uptake of undifferentiated thyroid cancer cells by adenovirus-mediated Na+/ I- symporter gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    So, Y.; Lee, Y. J.; Shin, J. H.; Oh, H. J.; Chung, J. K.; Lee, M. C.; Cho, B. Y. [College of Medicine, Univ. of Seoul National, Seoul (Korea, Republic of); Lee, K. H. [Samsung Medical Center, Seoul (Korea, Republic of)

    2003-07-01

    To increase radioiodine uptake on undifferentiated thyroid cancer cell (ARO cells) by adenovirus-mediated human Na+/I- symporter (hNIS) gene transfer. Recombinant adenovirus Ad-hNIS was manufactured successfully. After transfecting Ad-hNIS on ARO cells, in vitro I-125 uptake and efflux studies were performed. For in vivo studies, 1.510'8 p.f.u. (50 1) of Ad-hNIS was injected into xenograft ARO tumors on the R thigh of BALB/c nu/nu mice (n=12), and same amount of normal saline was injected into xenograft ARO tumors on the L thigh. Two, 3, 4 and 6 days after intratumoral injection of Ad-hNIS, I-131 images (3 mice per day) were taken and xenograft tumors on both thighs were all excised. Total RNA was extracted from each tumor tissue and RT-PCR was performed to confirm the hNIS expression of Ad-hNIS injected xenograft ARO tumors. I-125 uptake of Ad-hNIS transfected ARO cells was increased up to 233 folds at 120 minutes in vitro. I-125 efflux study revealed rapid washout of I-125 from Ad-hNIS transfected ARO cells. On dynamic image, I-131 uptake of Ad-hNIS injected ARO tumor was continuously increased until 60 minutes. Mean count ratios of xenograft ARO tumors (R/L) of 60 minutes I-131 images at 2, 3, 4 and 6 days after Ad-hNIS injection were 2.85, 2.54, 2.31, and 2.18, each. On RT-PCR, hNIS expression of Ad-hNIS transfected ARO xenograft tumors was confirmed. Radioiodine uptake was successfully increased in ARO cells by adenovirus-mediated hNIs gene transfer both in vitro and in vivo.

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

    Science.gov (United States)

    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.

  12. 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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

    International Nuclear Information System (INIS)

    Campos, Samuel K.; Barry, Michael A.

    2006-01-01

    The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  3. 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.

  4. Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

    Science.gov (United States)

    Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

    2000-01-01

    We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

  5. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang

    2013-01-01

    infection with lymphocytic choriomeningitis virus. We found that localized immunization with intermediate doses of Ad vector induce a moderate number of functional CD8 T cells, which qualitatively match those found in LCMV-infected mice. Numbers of these cells may be efficiently increased by additional...... adenoviral boosting and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad-vaccination will lead to even higher numbers of memory cells. In this case, the vaccination...

  6. Polarization transfer in (d-vector,n-vector) reactions

    International Nuclear Information System (INIS)

    Walter, R.L.; Tornow, W.

    1986-01-01

    The status of the measurements and the role of polarization transfer coefficients for (d/sup →/,n/sup →/) reactions is reviewed. Emphasis is given to reactions, involving light-nuclei systems. The importance of (d/sup →/,n/sup →/) reactions as sources of polarized neutrons is pointed out

  7. 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

  8. 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.

  9. [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.

  10. 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.

  11. 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

  12. Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

    Science.gov (United States)

    Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

    1995-10-01

    Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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

    Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

  15. Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

    International Nuclear Information System (INIS)

    Hwang, Kyung-Sun; Cho, Won-Kyung; Yoo, Jinsang; Yun, Hwan-Jung; Kim, Samyong; Im, Dong-Soo

    2005-01-01

    Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production. The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers

  16. 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.

    Science.gov (United States)

    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.

  17. 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

    Science.gov (United States)

    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 ...

  18. 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.

  19. 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.

  20. Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon

    Science.gov (United States)

    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

  1. 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.

    Directory of Open Access Journals (Sweden)

    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.

  2. 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...

  3. 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

  4. Adenovirus-mediated E2-EPF UCP gene transfer prevents autoamputation in a mouse model of hindlimb ischemia.

    Science.gov (United States)

    Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

    2012-04-01

    E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

  5. Linearized vector radiative transfer model MCC++ for a spherical atmosphere

    International Nuclear Information System (INIS)

    Postylyakov, O.V.

    2004-01-01

    Application of radiative transfer models has shown that optical remote sensing requires extra characteristics of radiance field in addition to the radiance intensity itself. Simulation of spectral measurements, analysis of retrieval errors and development of retrieval algorithms are in need of derivatives of radiance with respect to atmospheric constituents under investigation. The presented vector spherical radiative transfer model MCC++ was linearized, which allows the calculation of derivatives of all elements of the Stokes vector with respect to the volume absorption coefficient simultaneously with radiance calculation. The model MCC++ employs Monte Carlo algorithm for radiative transfer simulation and takes into account aerosol and molecular scattering, gas and aerosol absorption, and Lambertian surface albedo. The model treats a spherically symmetrical atmosphere. Relation of the estimated derivatives with other forms of radiance derivatives: the weighting functions used in gas retrieval and the air mass factors used in the DOAS retrieval algorithms, is obtained. Validation of the model against other radiative models is overviewed. The computing time of the intensity for the MCC++ model is about that for radiative models treating sphericity of the atmosphere approximately and is significantly shorter than that for the full spherical models used in the comparisons. The simultaneous calculation of all derivatives (i.e. with respect to absorption in all model atmosphere layers) and the intensity is only 1.2-2 times longer than the calculation of the intensity only

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

    Directory of Open Access Journals (Sweden)

    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.

  7. An efficient marker-free vector for clean gene transfer into plants ...

    African Journals Online (AJOL)

    A marker-free vector, pBINMF, for clean gene transfer was constructed based on the binary vector pBINPLUS. Vector pBINMF, carrying only a multiple cloning site (MCS) between the left and the right T-DNA border, was suitable to directly generate marker-free transgenic plants (MFTPs) without any vector sequences ...

  8. Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

    Science.gov (United States)

    Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

  9. 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.

  10. 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.

    Science.gov (United States)

    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.

  11. Use of adenoviral vectors as veterinary vaccines.

    Science.gov (United States)

    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.

  12. Resonance energy transfer: The unified theory via vector spherical harmonics

    Energy Technology Data Exchange (ETDEWEB)

    Grinter, Roger, E-mail: r.grinter@uea.ac.uk; Jones, Garth A., E-mail: garth.jones@uea.ac.uk [School of Chemistry, University of East Anglia, Norwich NR4 7TJ (United Kingdom)

    2016-08-21

    In this work, we derive the well-established expression for the quantum amplitude associated with the resonance energy transfer (RET) process between a pair of molecules that are beyond wavefunction overlap. The novelty of this work is that the field of the mediating photon is described in terms of a spherical wave rather than a plane wave. The angular components of the field are constructed in terms of vector spherical harmonics while Hankel functions are used to define the radial component. This approach alleviates the problem of having to select physically correct solution from non-physical solutions, which seems to be inherent in plane wave derivations. The spherical coordinate system allows one to easily decompose the photon’s fields into longitudinal and transverse components and offers a natural way to analyse near-, intermediate-, and far-zone RET within the context of the relative orientation of the transition dipole moments for the two molecules.

  13. Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

    Science.gov (United States)

    Harro, Clayton D; Robertson, Michael N; Lally, Michelle A; O'Neill, Lori D; Edupuganti, Srilatha; Goepfert, Paul A; Mulligan, Mark J; Priddy, Frances H; Dubey, Sheri A; Kierstead, Lisa S; Sun, Xiao; Casimiro, Danilo R; DiNubile, Mark J; Shiver, John W; Leavitt, Randi Y; Mehrotra, Devan V

    2009-01-01

    Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.

  14. Breadth of T cell responses after immunization with adenovirus vectors encoding ancestral antigens or polyvalent papillomavirus antigens

    DEFF Research Database (Denmark)

    Ragonnaud, Emeline; Pedersen, Anders Gorm; Holst, Peter Johannes

    2017-01-01

    to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...... circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did...

  15. Spectral element method for vector radiative transfer equation

    International Nuclear Information System (INIS)

    Zhao, J.M.; Liu, L.H.; Hsu, P.-F.; Tan, J.Y.

    2010-01-01

    A spectral element method (SEM) is developed to solve polarized radiative transfer in multidimensional participating medium. The angular discretization is based on the discrete-ordinates approach, and the spatial discretization is conducted by spectral element approach. Chebyshev polynomial is used to build basis function on each element. Four various test problems are taken as examples to verify the performance of the SEM. The effectiveness of the SEM is demonstrated. The h and the p convergence characteristics of the SEM are studied. The convergence rate of p-refinement follows the exponential decay trend and is superior to that of h-refinement. The accuracy and efficiency of the higher order approximation in the SEM is well demonstrated for the solution of the VRTE. The predicted angular distribution of brightness temperature and Stokes vector by the SEM agree very well with the benchmark solutions in references. Numerical results show that the SEM is accurate, flexible and effective to solve multidimensional polarized radiative transfer problems.

  16. Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

    Science.gov (United States)

    Geisbert, Joan B; Shedlock, Devon J; Xu, Ling; Lamoreaux, Laurie; Custers, Jerome H. H. V; Popernack, Paul M; Yang, Zhi-Yong; Pau, Maria G; Roederer, Mario; Koup, Richard A; Goudsmit, Jaap; Jahrling, Peter B; Nabel, Gary J

    2006-01-01

    Background Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. Methods and Findings To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously. Conclusions Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate. PMID:16683867

  17. Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs.

    Directory of Open Access Journals (Sweden)

    Nancy J Sullivan

    2006-06-01

    Full Text Available Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd encoding the Ebola glycoprotein (GP and nucleoprotein (NP has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10 particles, two logs lower than that used previously.Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.

  18. [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.

  19. 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

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

    Directory of Open Access Journals (Sweden)

    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

  1. Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis.

    Science.gov (United States)

    Santosuosso, Michael; McCormick, Sarah; Zhang, Xizhong; Zganiacz, Anna; Xing, Zhou

    2006-08-01

    Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. We examined a heterologous prime-boost regimen utilizing BCG as a prime vaccine and our recently described adenoviral vector expressing Ag85A (AdAg85A) as a boost vaccine. Since we recently demonstrated that a single intranasal but not intramuscular immunization with AdAg85A was able to induce potent protection from pulmonary Mycobacterium tuberculosis challenge in a mouse model, we compared the protective effects of parenteral and mucosal booster immunizations following subcutaneous BCG priming. Protection by BCG prime immunization was not effectively boosted by subcutaneous BCG or intramuscular AdAg85A. In contrast, protection by BCG priming was remarkably boosted by intranasal AdAg85A. Such enhanced protection by intranasal AdAg85A was correlated to the numbers of gamma interferon-positive CD4 and CD8 T cells residing in the airway lumen of the lung. Our study demonstrates that intranasal administration of AdAg85A represents an effective way to boost immune protection by parenteral BCG vaccination.

  2. 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

  3. 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.

  4. 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

    Objective: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene. herpes simplex virus type 1 thymidine kinase (HSV1-tk), were constructed and transferred into nee-natal cardiac myocytes, and a series of in vitro studies were carried out on the cells transferred to evaluate the uptake of radiolabeled reporter probe and to compare both vectors for cardiac reporter gene imaging. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSVI-tk. chosen as the reporter gene.was inserted into adenovirus vector (Ad5-tk) and plasmid (pDC316-tk), thus it could be transferred into neonatal cardiac myocytes. Recombinant adenovirus containing enhanced green fluorescent protein (Ad5-EGFP) was used as control. Recombinant plasmid was coated with lipofectamine TM 2000 (pDC316-tk/lipoplex). The specific reporter probe of HSV1-tk, 2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-uracil (FAU), was labeled with 131 I by solid phase oxidation with lodogen. Product wag purified on a reverse. phase Sep-Pak C18 column and the radiochemical purity wag then assessed. The accumulation of it in the transferred cardiac myocytes wag detected as uptake rate. Furthermore, mRNA expression of HSV1-tk was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while its protein expression wag located by immunocytochemistry. Results: FAU could be labeled with 131 I and the labeling efficiency was (53.82 ±2.05)%. The radiochemical purity was (94.85 ± 1.76)% after purification, and it kept stable in vitro for at least 24h. Time-dependent increase of the ac- cumulation of 131 I-FIAU was observed in both Ad5-tk group and pDC316-tk/lipoplex group. and the highest uptake rate occurred at 5h, with peak values of (12.55 ± 0.37)% and (2.09 ± 0.34)% respectively. However, it also indicated that greater

  5. 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.

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

    Science.gov (United States)

    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.

  7. 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

  8. Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine.

    Science.gov (United States)

    Cao, Yu; Liu, Zhenhai; Xie, Yilin; Hu, Jingchao; Wang, Hua; Fan, Zhipeng; Zhang, Chunmei; Wang, Jingsong; Wu, Chu-Tse; Wang, Songlin

    2015-12-15

    Periodontitis is one of the most widespread infectious diseases in humans. We previously promoted significant periodontal tissue regeneration in swine models with the transplantation of autologous periodontal ligament stem cells (PDLSCs) and PDLSC sheet. We also promoted periodontal tissue regeneration in a rat model with a local injection of allogeneic bone marrow mesenchymal stem cells. The purpose of the present study is to investigate the roles of the hepatocyte growth factor (HGF) and human dental pulp stem cells (DPSCs) in periodontal tissue regeneration in swine. In the present study, we transferred an adenovirus that carried HGF gene into human DPSCs (HGF-hDPSCs) under good manufacturing practice (GMP) conditions. These cells were then transplanted into a swine model for periodontal regeneration. Twenty miniature pigs were used to generate periodontitis with bone defect of 5 mm in width, 7 mm in length, and 3 mm in depth. After 12 weeks, clinical, radiological, quantitative and histological assessment of regenerated periodontal tissues was performed to compare periodontal regeneration in swine treated with cell implantation. Our study showed that injecting HGF-hDPSCs into this large animal model could significantly improve periodontal bone regeneration and soft tissue healing. A hDPSC or HGF-hDPSC sheet showed superior periodontal tissue regeneration compared to the injection of dissociated cells. However, the sheets required surgical placement; thus, they were suitable for surgically-managed periodontitis treatments. The adenovirus-mediated transfer of the HGF gene markedly decreased hDPSC apoptosis in a hypoxic environment or in serum-free medium, and it increased blood vessel regeneration. This study indicated that HGF-hDPSCs produced under GMP conditions significantly improved periodontal bone regeneration in swine; thus, this method represents a potential clinical application for periodontal regeneration.

  9. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

    OpenAIRE

    Podsakoff, G; Wong, K K; Chatterjee, S

    1994-01-01

    Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthe...

  10. 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

  11. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

    Science.gov (United States)

    Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

    2005-05-01

    Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

  12. Graphene materials as 2D non-viral gene transfer vector platforms.

    Science.gov (United States)

    Vincent, M; de Lázaro, I; Kostarelos, K

    2017-03-01

    Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.

  13. Highly efficient retrograde gene transfer into motor neurons by a lentiviral vector pseudotyped with fusion glycoprotein.

    Directory of Open Access Journals (Sweden)

    Miyabi Hirano

    Full Text Available The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G, permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet by pseudotyping a human immunodeficiency virus type 1 (HIV-1-based vector with fusion glycoprotein B type (FuG-B or a variant of FuG-B (FuG-B2, in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G. We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2 and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.

  14. Cancer gene therapy with targeted adenoviruses.

    Science.gov (United States)

    Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

    2008-11-01

    Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

  15. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

    Science.gov (United States)

    Podsakoff, G; Wong, K K; Chatterjee, S

    1994-09-01

    Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

  16. A new approach to radiative transfer theory using Jones's vectors. I

    International Nuclear Information System (INIS)

    Fymat, A.L.; Vasudevan, R.

    1975-01-01

    Radiative transfer of partially polarized radiation in an anisotropically scattering, inhomogeneous atmosphere containing arbitrary polydispersion of particles is described using Jones's amplitude vectors and matrices. This novel approach exploits the close analogy between the quantum mechanical states of spin 1/2 systems and the polarization states of electromagnetic radiation described by Jones's vector, and draws on the methodology of such spin 1/2 systems. The complete equivalence between the transport equation for Jones's vectors and the classical radiative transfer equation for Stokes's intensity vectors is demonstrated in two independent ways after deriving the transport equations for the polarization coherency matrices and for the quaternions corresponding to the Jones's vectors. A compact operator formulation of the theory is provided, and used to derive the necessary equations for both a local and a global description of the transport of Jones's vectors. Lastly, the integro-differential equations for the amplitude reflection and transmission matrices are derived, and related to the usual corresponding equations. The present formulation is the most succinct and the most convenient one for both theoretical and experimental studies. It yields a simpler analysis than the classical formulation since it reduces by a factor of two the dimensionality of transfer problems. It preserves information on phases, and thus can be used directly across the entire electromagnetic spectrum without any further conversion into intensities. (Auth.)

  17. 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

  18. 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.

  19. A new vector radiative transfer model as a part of SCIATRAN 3.0 software package.

    Science.gov (United States)

    Rozanov, Alexei; Rozanov, Vladimir; Burrows, John P.

    The SCIATRAN 3.0 package is a result of further development of the SCIATRAN 2.x software family which, similar to previous versions, comprises a radiative transfer model and a retrieval block. A major improvement was achieved in comparison to previous software versions by adding the vector mode to the radiative transfer model. Thus, the well-established Discrete Ordinate solver can now be run in the vector mode to calculate the scattered solar radiation including polarization, i.e., to simulate all four components of the Stockes vector. Similar to the scalar version, the simulations can be performed for any viewing geometry typical for atmospheric observations in the UV-Vis-NIR spectral range (nadir, limb, off-axis, etc.) as well as for any observer position within or outside the Earth's atmosphere. Similar to the precursor version, the new model is freely available for non-commercial use via the web page of the University of Bremen. In this presentation a short description of the software package, especially of the new vector radiative transfer model will be given, including remarks on the availability for the scientific community. Furthermore, comparisons to other vector models will be shown and some example problems will be considered where the polarization of the observed radiation must be accounted for to obtain high quality results.

  20. A passive and active microwave-vector radiative transfer (PAM-VRT) model

    International Nuclear Information System (INIS)

    Yang, Jun; Min, Qilong

    2015-01-01

    A passive and active microwave vector radiative transfer (PAM-VRT) package has been developed. This fast and accurate forward microwave model, with flexible and versatile input and output components, self-consistently and realistically simulates measurements/radiation of passive and active microwave sensors. The core PAM-VRT, microwave radiative transfer model, consists of five modules: gas absorption (two line-by-line databases and four fast models); hydrometeor property of water droplets and ice (spherical and nonspherical) particles; surface emissivity (from Community Radiative Transfer Model (CRTM)); vector radiative transfer of successive order of scattering (VSOS); and passive and active microwave simulation. The PAM-VRT package has been validated against other existing models, demonstrating good accuracy. The PAM-VRT not only can be used to simulate or assimilate measurements of existing microwave sensors, but also can be used to simulate observation results at some new microwave sensors. - Highlights: • A novel microwave vector radiative transfer model is developed. • It can simulate passive and active microwave radiative transfer simultaneously. • It can be applied to simulate measurements for different types of viewing geometry. • The accuracy of this model has been validated against other existing models

  1. 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.

  2. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined......This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...

  3. On the derivation of vector radiative transfer equation for polarized radiative transport in graded index media

    International Nuclear Information System (INIS)

    Zhao, J.M.; Tan, J.Y.; Liu, L.H.

    2012-01-01

    Light transport in graded index media follows a curved trajectory determined by Fermat's principle. Besides the effect of variation of the refractive index on the transport of radiative intensity, the curved ray trajectory will induce geometrical effects on the transport of polarization ellipse. This paper presents a complete derivation of vector radiative transfer equation for polarized radiation transport in absorption, emission and scattering graded index media. The derivation is based on the analysis of the conserved quantities for polarized light transport along curved trajectory and a novel approach. The obtained transfer equation can be considered as a generalization of the classic vector radiative transfer equation that is only valid for uniform refractive index media. Several variant forms of the transport equation are also presented, which include the form for Stokes parameters defined with a fixed reference and the Eulerian forms in the ray coordinate and in several common orthogonal coordinate systems.

  4. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes

    2012-01-01

    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 CD......8 T-cell memory even in individuals with pre-existing vector immunity....

  5. Development of replication-deficient adenovirus malaria vaccines.

    Science.gov (United States)

    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.

  6. 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.

    Directory of Open Access Journals (Sweden)

    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

  7. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    Science.gov (United States)

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  8. The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Cao, Lei; Yang, Fei; Liu, Guangwang; Yu, Degang; Li, Huiwu; Fan, Qiming; Gan, Yaokai; Tang, Tingting; Dai, Kerong

    2011-06-01

    Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. A successive order of scattering model for solving vector radiative transfer in the atmosphere

    International Nuclear Information System (INIS)

    Min Qilong; Duan Minzheng

    2004-01-01

    A full vector radiative transfer model for vertically inhomogeneous plane-parallel media has been developed by using the successive order of scattering approach. In this model, a fast analytical expansion of Fourier decomposition is implemented and an exponent-linear assumption is used for vertical integration. An analytic angular interpolation method of post-processing source function is also implemented to accurately interpolate the Stokes vector at arbitrary angles for a given solution. It has been tested against the benchmarks for the case of randomly orientated oblate spheroids, illustrating a good agreement for each stokes vector (within 0.01%). Sensitivity tests have been conducted to illustrate the accuracy of vertical integration and angle interpolation approaches. The contribution of each scattering order for different optical depths and single scattering albedos are also analyzed

  10. Microplastics as Vectors for Environmental Contaminants : Exploring Sorption, Desorption, and Transfer to Biota

    DEFF Research Database (Denmark)

    Hartmann, Nanna B.; Rist, Sinja; Bodin, Julia

    2017-01-01

    The occurrence and effects of microplastics (MPs) in the aquatic environment are receiving increasing attention. In addition to their possible direct adverse effects on biota, the potential role of MPs as vectors for hydrophobic organic chemicals (HOCs), compared to natural pathways, is a topic...... outline research needed to fill knowledge gaps and improve model-based calculations of MP-facilitated HOC transfer in the environment. Integr Environ Assess Manag 2017;13:488–493. © 2017 SETAC...... of much debate. It is evident, however, that temporal and spatial variations of MP occurrence do (and will) occur. To further improve the estimations of the role of MPs as vectors for HOC transfer into biota under varying MP concentrations and environmental conditions, it is important to identify...

  11. The effects of combining ionizing radiation and adenovirus-mediated p53 gene transfer in human nasopharyngeal carcinoma cell lines

    International Nuclear Information System (INIS)

    Liu Feifei; Li Jianhua; Lax, Stuart; Klamut, Henry

    1997-01-01

    Purpose/Objective: We have previously demonstrated that the introduction of human recombinant wild-type p53 carried by the adenoviral vector (Ad5CMV-p53) into two human nasopharyngeal carcinoma (NPC) cell lines (CNE-1 and CNE-2Z) resulted in significant cytotoxicity. In the current work, we wanted to evaluate the results of this strategy when combined with ionizing radiation (XRT). Materials and Methods: CNE-1, CNE-2Z, and a normal human nasopharyngeal fibroblast strain KS1, were infected with iso-effective doses of 2, 6 and 6 pfu/cell of Ad5CMV-p53 respectively. XRT was administered 24 hours post-infection, to coincide with the time of maximal recombinant p53 expression. Western blot analyses were conducted for p53, p21 WAF1/CIP1 , bax and bcl-2. Cell viability was evaluated using both the MTT and clonogenic assays. Presence of apoptosis was determined by using DNA agarose gel electrophoresis. Results: We observed that the combination of Ad5CMV-p53 + XRT (2, 4, and 6 Gy) resulted in an approximately 1-log greater level of cytotoxicity compared to that observed with XRT alone for both NPC cell lines. The MTT assay indicated sparing of the KS1 cells when subjected to the identical treatments. XRT alone stimulated minimal p53 expression; Ad5CMV-p53 alone induced significant recombinant p53 expression, which was not further enhanced by the addition of XRT. Similar observations were made for p21 WAF1/CIP1 expression. No changes were observed for bax and bcl-2 expression with any of these treatments. Apoptosis was induced following 4 Gy of XRT alone, but was observed earlier, at 2 Gy when combined with Ad5CMV-p53. Conclusion: Additional cytotoxicity was observed for the NPC cell lines when XRT was combined with Ad5CMV-p53 infection, with concurrent sparing of normal cells (KS1). This cytotoxicity also appeared to be mediated through the induction of the apoptotic pathway. These results support our previous observation of the potential application of this strategy in the

  12. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid, Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

    OpenAIRE

    Casimiro, Danilo R.; Chen, Ling; Fu, Tong-Ming; Evans, Robert K.; Caulfield, Michael J.; Davies, Mary-Ellen; Tang, Aimin; Chen, Minchun; Huang, Lingyi; Harris, Virginia; Freed, Daniel C.; Wilson, Keith A.; Dubey, Sheri; Zhu, De-Min; Nawrocki, Denise

    2003-01-01

    Cellular immune responses, particularly those associated with CD3+ CD8+ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defecti...

  13. Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

    OpenAIRE

    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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. Transient gene transfer to neurons and glia : analysis of adenoviral vector performance in the CNS and PNS

    NARCIS (Netherlands)

    Hermens, W.T.J.M.C.; Giger, Roman J; Holtmaat, Anthony J D G; Dijkhuizen, Paul A; Houweling, D A; Verhaagen, J

    In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied,

  17. Adenoviral vector-mediated gene transfer and neurotransplantation : possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus

    NARCIS (Netherlands)

    van Esseveldt, K E; Liu, R.; Hermens, W.T.J.M.C.; Verhaagen, J; Boer, G J

    Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural

  18. T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

    Science.gov (United States)

    Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

    2014-05-01

    Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

  19. Gene transfer strategies for improving radiolabeled peptide imaging and therapy

    International Nuclear Information System (INIS)

    Rogers, B.E.; Buchsbaum, D.J.; Zinn, K.R.

    2000-01-01

    Utilization of molecular biology techniques offers attractive options in nuclear medicine for improving cancer imaging and therapy with radiolabeled peptides. Two of these options include utilization of phage-panning to identify novel tumor specific peptides or single chain antibodies and gene transfer techniques to increase the antibodies and gene transfer techniques to increase the number of antigen/receptor sites expressed on malignant cells. The group has focused on the latter approach for improving radiolabeled peptide imaging and therapy. The most widely used gene transfer vectors in clinical gene therapy trials include retrovirus, cationic lipids and adenovirus. It has been utilized adenovirus vectors for gene transfer because of their ability to accomplish efficient in vivo gene transfer. Adenovirus vectors encoding the genes for a variety of antigens/receptors (carcinoembryonic antigen, gastrin-releasing peptide receptor, somatostatin receptor subtype 2 (SSTr2) have all shown that their expression is increased on cancer cells both in vitro and in vivo following adenovirus infection. Of particular interest has been the adenovirus encoding for SSTr2 (AdCMVSSTr2). Various radioisotopes have been attached to somatostatin analogues for imaging and therapy of SSTr2-positive tumors both clinically and in animal models. The use of these analogues in combination with AdCMVSSTr2 is a promising approach for improving the detection sensitivity and therapeutic efficacy of these radiolabeled peptides against solid tumors. In addition, it has been proposed the use of SSTr2 as a marker for imaging the expression of another cancer therapeutic transgene (e.g. cytosine deaminase, thymidine kinase) encoded within the same vector. This would allow for non-invasive monitoring of gene delivery to tumor sites

  20. Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial

    Directory of Open Access Journals (Sweden)

    Chongsheng Qian

    2017-05-01

    Full Text Available Abstract Background Allogeneic hematopoietic stem cell transplantation (HSCT, the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB. Methods Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9% or a third party haploidentical donor (57.1%. One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. Results One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%. Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. Conclusions Adoptive transfer of rapidly isolated ADV

  1. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    Science.gov (United States)

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

  2. Geodesic Flow Kernel Support Vector Machine for Hyperspectral Image Classification by Unsupervised Subspace Feature Transfer

    Directory of Open Access Journals (Sweden)

    Alim Samat

    2016-03-01

    Full Text Available In order to deal with scenarios where the training data, used to deduce a model, and the validation data have different statistical distributions, we study the problem of transformed subspace feature transfer for domain adaptation (DA in the context of hyperspectral image classification via a geodesic Gaussian flow kernel based support vector machine (GFKSVM. To show the superior performance of the proposed approach, conventional support vector machines (SVMs and state-of-the-art DA algorithms, including information-theoretical learning of discriminative cluster for domain adaptation (ITLDC, joint distribution adaptation (JDA, and joint transfer matching (JTM, are also considered. Additionally, unsupervised linear and nonlinear subspace feature transfer techniques including principal component analysis (PCA, randomized nonlinear principal component analysis (rPCA, factor analysis (FA and non-negative matrix factorization (NNMF are investigated and compared. Experiments on two real hyperspectral images show the cross-image classification performances of the GFKSVM, confirming its effectiveness and suitability when applied to hyperspectral images.

  3. Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

    Directory of Open Access Journals (Sweden)

    Lauro Augusto de Oliveira

    2010-10-01

    Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

  4. 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

  5. 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.

  6. Poynting vector analysis for wireless power transfer between magnetically coupled coils with different loads.

    Science.gov (United States)

    Guo, Yunsheng; Li, Jiansheng; Hou, Xiaojuan; Lv, Xiaolong; Liang, Hao; Zhou, Ji; Wu, Hongya

    2017-04-07

    Wireless power transfer is a nonradiative type of transmission that is performed in the near-field region. In this region, the electromagnetic fields that are produced by both the transmitting and receiving coils are evanescent fields, which should not transmit energy. This then raises the question of how the energy can be transferred. Here we describe a theoretical study of the two evanescent field distributions at different terminal loads. It is shown that the essential principle of wireless energy transfer is the superposition of the two evanescent fields, and the resulting superimposed field is mediated through the terminal load. If the terminal load is either capacitive or inductive, then the superimposed field cannot transfer the energy because its Poynting vector is zero; in contrast, if the load is resistive, energy can then be conveyed from the transmitting coil to the receiving coil. The simulation results for the magnetic field distributions and the time-domain current waveforms agree very well with the results of the theoretical analysis. This work thus provides a comprehensive understanding of the energy transfer mechanism involved in the magnetic resonant coupling system.

  7. Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

    Science.gov (United States)

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I.; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-01-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options. PMID:24925206

  8. Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

    Science.gov (United States)

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-10-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

  9. Interpreting angular momentum transfer between electromagnetic multipoles using vector spherical harmonics.

    Science.gov (United States)

    Grinter, Roger; Jones, Garth A

    2018-02-01

    The transfer of angular momentum between a quadrupole emitter and a dipole acceptor is investigated theoretically. Vector spherical harmonics are used to describe the angular part of the field of the mediating photon. Analytical results are presented for predicting angular momentum transfer between the emitter and absorber within a quantum electrodynamical framework. We interpret the allowability of such a process, which appears to violate conservation of angular momentum, in terms of the breakdown of the isotropy of space at the point of photon absorption (detection). That is, collapse of the wavefunction results in loss of all angular momentum information. This is consistent with Noether's Theorem and demystifies some common misconceptions about the nature of the photon. The results have implications for interpreting the detection of photons from multipole sources and offers insight into limits on information that can be extracted from quantum measurements in photonic systems.

  10. Vector Green's function algorithm for radiative transfer in plane-parallel atmosphere

    Energy Technology Data Exchange (ETDEWEB)

    Qin Yi [School of Physics, University of New South Wales (Australia)]. E-mail: yi.qin@csiro.au; Box, Michael A. [School of Physics, University of New South Wales (Australia)

    2006-01-15

    Green's function is a widely used approach for boundary value problems. In problems related to radiative transfer, Green's function has been found to be useful in land, ocean and atmosphere remote sensing. It is also a key element in higher order perturbation theory. This paper presents an explicit expression of the Green's function, in terms of the source and radiation field variables, for a plane-parallel atmosphere with either vacuum boundaries or a reflecting (BRDF) surface. Full polarization state is considered but the algorithm has been developed in such way that it can be easily reduced to solve scalar radiative transfer problems, which makes it possible to implement a single set of code for computing both the scalar and the vector Green's function.

  11. Vector Green's function algorithm for radiative transfer in plane-parallel atmosphere

    International Nuclear Information System (INIS)

    Qin Yi; Box, Michael A.

    2006-01-01

    Green's function is a widely used approach for boundary value problems. In problems related to radiative transfer, Green's function has been found to be useful in land, ocean and atmosphere remote sensing. It is also a key element in higher order perturbation theory. This paper presents an explicit expression of the Green's function, in terms of the source and radiation field variables, for a plane-parallel atmosphere with either vacuum boundaries or a reflecting (BRDF) surface. Full polarization state is considered but the algorithm has been developed in such way that it can be easily reduced to solve scalar radiative transfer problems, which makes it possible to implement a single set of code for computing both the scalar and the vector Green's function

  12. 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

  13. Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  15. Acoustical contribution calculation and analysis of compressor shell based on acoustic transfer vector method

    Science.gov (United States)

    Chen, Xiaol; Guo, Bei; Tuo, Jinliang; Zhou, Ruixin; Lu, Yang

    2017-08-01

    Nowadays, people are paying more and more attention to the noise reduction of household refrigerator compressor. This paper established a sound field bounded by compressor shell and ISO3744 standard field points. The Acoustic Transfer Vector (ATV) in the sound field radiated by a refrigerator compressor shell were calculated which fits the test result preferably. Then the compressor shell surface is divided into several parts. Based on Acoustic Transfer Vector approach, the sound pressure contribution to the field points and the sound power contribution to the sound field of each part were calculated. To obtain the noise radiation in the sound field, the sound pressure cloud charts were analyzed, and the contribution curves in different frequency of each part were acquired. Meanwhile, the sound power contribution of each part in different frequency was analyzed, to ensure those parts where contributes larger sound power. Through the analysis of acoustic contribution, those parts where radiate larger noise on the compressor shell were determined. This paper provides a credible and effective approach on the structure optimal design of refrigerator compressor shell, which is meaningful in the noise and vibration reduction.

  16. A glycolipid adjuvant, 7DW8-5, enhances CD8+ T cell responses induced by an adenovirus-vectored malaria vaccine in non-human primates.

    Science.gov (United States)

    Padte, Neal N; Boente-Carrera, Mar; Andrews, Chasity D; McManus, Jenny; Grasperge, Brooke F; Gettie, Agegnehu; Coelho-dos-Reis, Jordana G; Li, Xiangming; Wu, Douglass; Bruder, Joseph T; Sedegah, Martha; Patterson, Noelle; Richie, Thomas L; Wong, Chi-Huey; Ho, David D; Vasan, Sandhya; Tsuji, Moriya

    2013-01-01

    A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.

  17. A glycolipid adjuvant, 7DW8-5, enhances CD8+ T cell responses induced by an adenovirus-vectored malaria vaccine in non-human primates.

    Directory of Open Access Journals (Sweden)

    Neal N Padte

    Full Text Available A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer, enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA, consists of two non-replicating recombinant adenoviral (Ad vectors, one expressing the circumsporozoite protein (CSP and another expressing the apical membrane antigen-1 (AMA1 of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.

  18. A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

    Science.gov (United States)

    Fu, Yuan-Hui; He, Jin-Sheng; Wang, Xiao-Bo; Zheng, Xian-Xian; Wu, Qiang; Xie, Can; Zhang, Mei; Wei, Wei; Tang, Qian; Song, Jing-Dong; Qu, Jian-Guo; Hong, Tao

    2010-04-23

    Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice. 2010 Elsevier Inc. All rights reserved.

  19. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

    Directory of Open Access Journals (Sweden)

    Gideon Hen

    Full Text Available The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV, into the chorioallantoic membrane (CAM of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP or recombinant alpha-melanocyte-stimulating hormone (α-MSH genes, driven by the cytomegalovirus (CMV promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP nick end labeling (TUNEL assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA, and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

  20. Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

    Science.gov (United States)

    Vink, Conrad A; Counsell, John R; Perocheau, Dany P; Karda, Rajvinder; Buckley, Suzanne M K; Brugman, Martijn H; Galla, Melanie; Schambach, Axel; McKay, Tristan R; Waddington, Simon N; Howe, Steven J

    2017-08-02

    Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. vSmartMOM: A vector matrix operator method-based radiative transfer model linearized with respect to aerosol properties

    International Nuclear Information System (INIS)

    Sanghavi, Suniti; Davis, Anthony B.; Eldering, Annmarie

    2014-01-01

    In this paper, we build up on the scalar model smartMOM to arrive at a formalism for linearized vector radiative transfer based on the matrix operator method (vSmartMOM). Improvements have been made with respect to smartMOM in that a novel method of computing intensities for the exact viewing geometry (direct raytracing) without interpolation between quadrature points has been implemented. Also, the truncation method employed for dealing with highly peaked phase functions has been changed to a vector adaptation of Wiscombe's delta-m method. These changes enable speedier and more accurate radiative transfer computations by eliminating the need for a large number of quadrature points and coefficients for generalized spherical functions. We verify our forward model against the benchmarking results of Kokhanovsky et al. (2010) [22]. All non-zero Stokes vector elements are found to show agreement up to mostly the seventh significant digit for the Rayleigh atmosphere. Intensity computations for aerosol and cloud show an agreement of well below 0.03% and 0.05% at all viewing angles except around the solar zenith angle (60°), where most radiative models demonstrate larger variances due to the strongly forward-peaked phase function. We have for the first time linearized vector radiative transfer based on the matrix operator method with respect to aerosol optical and microphysical parameters. We demonstrate this linearization by computing Jacobian matrices for all Stokes vector elements for a multi-angular and multispectral measurement setup. We use these Jacobians to compare the aerosol information content of measurements using only the total intensity component against those using the idealized measurements of full Stokes vector [I,Q,U,V] as well as the more practical use of only [I,Q,U]. As expected, we find for the considered example that the accuracy of the retrieved parameters improves when the full Stokes vector is used. The information content for the full Stokes

  2. Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

    Science.gov (United States)

    Buttgereit, P; Weineck, S; Röpke, G; Märten, A; Brand, K; Heinicke, T; Caselmann, W H; Huhn, D; Schmidt-Wolf, I G

    2000-08-01

    Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and

  3. 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...

  4. Adenovirus (For Parents)

    Science.gov (United States)

    ... 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 ...

  5. 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.

  6. Modeling horizontal gene transfer (HGT in the gut of the Chagas disease vector Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Durvasula Ravi V

    2011-05-01

    Full Text Available Abstract Background Paratransgenesis is an approach to reducing arthropod vector competence using genetically modified symbionts. When applied to control of Chagas disease, the symbiont bacterium Rhodococcus rhodnii, resident in the gut lumen of the triatomine vector Rhodnius prolixus (Hemiptera: Reduviidae, is transformed to export cecropin A, an insect immune peptide. Cecropin A is active against Trypanosoma cruzi, the causative agent of Chagas disease. While proof of concept has been achieved in laboratory studies, a rigorous and comprehensive risk assessment is required prior to consideration of field release. An important part of this assessment involves estimating probability of transgene horizontal transfer to environmental organisms (HGT. This article presents a two-part risk assessment methodology: a theoretical model predicting HGT in the gut of R. prolixus from the genetically transformed symbiont R. rhodnii to a closely related non-target bacterium, Gordona rubropertinctus, in the absence of selection pressure, and a series of laboratory trials designed to test the model. Results The model predicted an HGT frequency of less than 1.14 × 10-16 per 100,000 generations at the 99% certainty level. The model was iterated twenty times, with the mean of the ten highest outputs evaluated at the 99% certainty level. Laboratory trials indicated no horizontal gene transfer, supporting the conclusions of the model. Conclusions The model treats HGT as a composite event, the probability of which is determined by the joint probability of three independent events: gene transfer through the modalities of transformation, transduction, and conjugation. Genes are represented in matrices and Monte Carlo method and Markov chain analysis are used to simulate and evaluate environmental conditions. The model is intended as a risk assessment instrument and predicts HGT frequency of less than 1.14 × 10-16 per 100,000 generations. With laboratory studies that

  7. Polarimetric signatures of a canopy of dielectric cylinders based on first and second order vector radiative transfer theory

    Science.gov (United States)

    Tsang, Leung; Chan, Chi Hou; Kong, Jin AU; Joseph, James

    1992-01-01

    Complete polarimetric signatures of a canopy of dielectric cylinders overlying a homogeneous half space are studied with the first and second order solutions of the vector radiative transfer theory. The vector radiative transfer equations contain a general nondiagonal extinction matrix and a phase matrix. The energy conservation issue is addressed by calculating the elements of the extinction matrix and the elements of the phase matrix in a manner that is consistent with energy conservation. Two methods are used. In the first method, the surface fields and the internal fields of the dielectric cylinder are calculated by using the fields of an infinite cylinder. The phase matrix is calculated and the extinction matrix is calculated by summing the absorption and scattering to ensure energy conservation. In the second method, the method of moments is used to calculate the elements of the extinction and phase matrices. The Mueller matrix based on the first order and second order multiple scattering solutions of the vector radiative transfer equation are calculated. Results from the two methods are compared. The vector radiative transfer equations, combined with the solution based on method of moments, obey both energy conservation and reciprocity. The polarimetric signatures, copolarized and depolarized return, degree of polarization, and phase differences are studied as a function of the orientation, sizes, and dielectric properties of the cylinders. It is shown that second order scattering is generally important for vegetation canopy at C band and can be important at L band for some cases.

  8. Multiple scattering effects with cyclical terms in active remote sensing of vegetated surface using vector radiative transfer theory

    Science.gov (United States)

    The energy transport in a vegetated (corn) surface layer is examined by solving the vector radiative transfer equation using a numerical iterative approach. This approach allows a higher order that includes the multiple scattering effects. Multiple scattering effects are important when the optical t...

  9. 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.

  10. 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

  11. A vector radiative transfer model for coupled atmosphere and ocean systems with a rough interface

    International Nuclear Information System (INIS)

    Zhai Pengwang; Hu Yongxiang; Chowdhary, Jacek; Trepte, Charles R.; Lucker, Patricia L.; Josset, Damien B.

    2010-01-01

    We report on an exact vector (polarized) radiative transfer (VRT) model for coupled atmosphere and ocean systems. This VRT model is based on the successive order of scattering (SOS) method, which virtually takes all the multiple scattering processes into account, including atmospheric scattering, oceanic scattering, reflection and transmission through the rough ocean surface. The isotropic Cox-Munk wave model is used to derive the ref and transmission matrices for the rough ocean surface. Shadowing effects are included by the shadowing function. We validated the SOS results by comparing them with those calculated by two independent codes based on the doubling/adding and Monte Carlo methods. Two error analyses related to the ocean color remote sensing are performed in the coupled atmosphere and ocean systems. One is the scalar error caused by ignoring the polarization in the whole system. The other is the error introduced by ignoring the polarization of the light transmitted through the ocean interface. Both errors are significant for the cases studied. This code fits for the next generation of ocean color study because it converges fast for absorbing medium as, for instance, ocean.

  12. Considering polarization in MODIS-based cloud property retrievals by using a vector radiative transfer code

    International Nuclear Information System (INIS)

    Yi, Bingqi; Huang, Xin; Yang, Ping; Baum, Bryan A.; Kattawar, George W.

    2014-01-01

    In this study, a full-vector, adding–doubling radiative transfer model is used to investigate the influence of the polarization state on cloud property retrievals from Moderate Resolution Imaging Spectroradiometer (MODIS) satellite observations. Two sets of lookup tables (LUTs) are developed for the retrieval purposes, both of which provide water cloud and ice cloud reflectivity functions at two wavelengths in various sun-satellite viewing geometries. However, only one of the LUTs considers polarization. The MODIS reflectivity observations at 0.65 μm (band 1) and 2.13 μm (band 7) are used to infer the cloud optical thickness and particle effective diameter, respectively. Results indicate that the retrievals for both water cloud and ice cloud show considerable sensitivity to polarization. The retrieved water and ice cloud effective diameter and optical thickness differences can vary by as much as ±15% due to polarization state considerations. In particular, the polarization state has more influence on completely smooth ice particles than on severely roughened ice particles. - Highlights: • Impact of polarization on satellite-based retrieval of water/ice cloud properties is studied. • Inclusion of polarization can change water/ice optical thickness and effective diameter values by up to ±15%. • Influence of polarization on cloud property retrievals depends on sun-satellite viewing geometries

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  15. 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.

    Directory of Open Access Journals (Sweden)

    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

  16. 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.

    Science.gov (United States)

    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

  17. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Adaptation of the delta-m and δ-fit truncation methods to vector radiative transfer: Effect of truncation on radiative transfer accuracy

    International Nuclear Information System (INIS)

    Sanghavi, Suniti; Stephens, Graeme

    2015-01-01

    In the presence of aerosol and/or clouds, the use of appropriate truncation methods becomes indispensable for accurate but cost-efficient radiative transfer computations. Truncation methods allow the reduction of the large number (usually several hundreds) of Fourier components associated with particulate scattering functions to a more manageable number, thereby making it possible to carry out radiative transfer computations with a modest number of streams. While several truncation methods have been discussed for scalar radiative transfer, few rigorous studies have been made of truncation methods for the vector case. Here, we formally derive the vector form of Wiscombe's delta-m truncation method. Two main sources of error associated with delta-m truncation are identified as the delta-separation error (DSE) and the phase-truncation error (PTE). The view angles most affected by truncation error occur in the vicinity of the direction of exact backscatter. This view geometry occurs commonly in satellite based remote sensing applications, and is hence of considerable importance. In order to deal with these errors, we adapt the δ-fit approach of Hu et al. (2000) [17] to vector radiative transfer. The resulting δBGE-fit is compared with the vectorized delta-m method. For truncation at l=25 of an original phase matrix consisting of over 300 Fourier components, the use of the δBGE-fit minimizes the error due to truncation at these view angles, while practically eliminating error at other angles. We also show how truncation errors have a distorting effect on hyperspectral absorption line shapes. The choice of the δBGE-fit method over delta-m truncation minimizes errors in absorption line depths, thus affording greater accuracy for sensitive retrievals such as those of XCO 2 from OCO-2 or GOSAT measurements. - Highlights: • Derives vector form for delta-m truncation method. • Adapts δ-fit truncation approach to vector RTE as δBGE-fit. • Compares truncation

  20. 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

  1. An efficient marker-free vector for clean gene transfer into plants

    African Journals Online (AJOL)

    Yomi

    2012-02-23

    Feb 23, 2012 ... vector sequences inserted into plant genomes. Transformation ..... Thymosin Alpha 1 (Tα1) in Marker-free Transgenic Lettuce (Lactuca sativa). ... obtaining marker-free plants of a cross-pollinating and vegetatively propagated ...

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier.

    Science.gov (United States)

    Purdy, Des; O'Keeffe, Triona A T; Elmore, Michael; Herbert, Mike; McLeod, Anne; Bokori-Brown, Monika; Ostrowski, Anna; Minton, Nigel P

    2002-10-01

    Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT-based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5'-GGNMCC-3') and MboI/M. MboI (5'-GMATC-3') respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5'-CATCG-3' between the fourth and fifth nucleotide to give a blunt-ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five- and 16-fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 x 10-6 and 5.5 x 10-5 transconjugants per donor.

  7. Ethical considerations in the use of lentiviral vectors for genetic transfer.

    Science.gov (United States)

    Roy, I

    2001-11-01

    This chapter will outline the various concerns which have been raised in scientific, bioethics, and lay communities about the use of lentiviral vectors for purposes of gene therapy. Many of these concerns are ranged around gene therapy itself; others are concerns particular to using this sort of vector for genetic modification of human cells. These concerns are outlined within the chapter, and arguments are given in favor and against various approaches to these concerns. Lastly, it is noted throughout that at this stage of research into gene therapy, the most practical approach to these dilemmas is to maintain awareness of the ethical problems and provide information to those concerned with all aspects of the development of this set of technologies.

  8. Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

    Science.gov (United States)

    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.

  9. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. 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

  11. 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)

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

    Directory of Open Access Journals (Sweden)

    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

  13. Determining the effect of solid and liquid vectors on the gaseous interfacial area and oxygen transfer rates in two-phase partitioning bioreactors

    International Nuclear Information System (INIS)

    Quijano, Guillermo; Rocha-Rios, Jose; Hernandez, Maria; Villaverde, Santiago; Revah, Sergio; Munoz, Raul; Thalasso, Frederic

    2010-01-01

    The effect of liquid and solid transfer vectors (silicone oil and Desmopan, respectively) on the gaseous interfacial area (a g ) was evaluated in a two-phase partitioning bioreactor (TPPB) using fresh mineral salt medium and the cultivation broth of a toluene degradation culture (Pseudomonas putida DOT-T1E cultures continuously cultivated with and without silicone oil at low toluene loading rates). Higher values of a g were recorded in the presence of both silicone oil and Desmopan compared to the values obtained in the absence of a vector, regardless of the aqueous medium tested (1.6 and 3 times higher, respectively, using fresh mineral salt medium). These improvements in a g were well correlated to the oxygen mass transfer enhancements supported by the vectors (1.3 and 2.5 for liquid and solid vectors, respectively, using fresh medium). In this context, oxygen transfer rates of 2.5 g O 2 L -1 h -1 and 1.3 g O 2 L -1 h -1 were recorded in the presence of Desmopan and silicone oil, respectively, which are in agreement with previously reported values in literature. These results suggest that mass transfer enhancements in TPPBs might correspond to an increase in a g rather than to the establishment of a high-performance gas/vector/water transfer pathway.

  14. Determining the effect of solid and liquid vectors on the gaseous interfacial area and oxygen transfer rates in two-phase partitioning bioreactors

    Energy Technology Data Exchange (ETDEWEB)

    Quijano, Guillermo [Departmento de Ingenieria Quimica y Tecnologia del Medio Ambiente, Universidad de Valladolid, Paseo del Prado de la Magdalena, s/n, 47005 Valladolid (Spain); Departamento de Biotecnologia y Bioingenieria, Centro de Investigacion y de Estudios, Avanzados del IPN (Cinvestav), Apdo. Postal 14-740, 07360 Mexico, D.F. (Mexico); Rocha-Rios, Jose [Departmento de Ingenieria Quimica y Tecnologia del Medio Ambiente, Universidad de Valladolid, Paseo del Prado de la Magdalena, s/n, 47005 Valladolid (Spain); Departamento de Ingenieria de Procesos e Hidraulica (IPH), Universidad Autonoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, 09340 Mexico, D.F. (Mexico); Hernandez, Maria; Villaverde, Santiago [Departmento de Ingenieria Quimica y Tecnologia del Medio Ambiente, Universidad de Valladolid, Paseo del Prado de la Magdalena, s/n, 47005 Valladolid (Spain); Revah, Sergio [Departamento de Procesos y Tecnologia, Universidad Autonoma Metropolitana-Cuajimalpa, c/o IPH, UAM-Iztapalapa, Av. San Rafael Atlixco No. 186, 09340 Mexico, D.F. (Mexico); Munoz, Raul, E-mail: mutora@iq.uva.es [Departmento de Ingenieria Quimica y Tecnologia del Medio Ambiente, Universidad de Valladolid, Paseo del Prado de la Magdalena, s/n, 47005 Valladolid (Spain); Thalasso, Frederic [Departamento de Biotecnologia y Bioingenieria, Centro de Investigacion y de Estudios, Avanzados del IPN (Cinvestav), Apdo. Postal 14-740, 07360 Mexico, D.F. (Mexico)

    2010-03-15

    The effect of liquid and solid transfer vectors (silicone oil and Desmopan, respectively) on the gaseous interfacial area (a{sub g}) was evaluated in a two-phase partitioning bioreactor (TPPB) using fresh mineral salt medium and the cultivation broth of a toluene degradation culture (Pseudomonas putida DOT-T1E cultures continuously cultivated with and without silicone oil at low toluene loading rates). Higher values of a{sub g} were recorded in the presence of both silicone oil and Desmopan compared to the values obtained in the absence of a vector, regardless of the aqueous medium tested (1.6 and 3 times higher, respectively, using fresh mineral salt medium). These improvements in a{sub g} were well correlated to the oxygen mass transfer enhancements supported by the vectors (1.3 and 2.5 for liquid and solid vectors, respectively, using fresh medium). In this context, oxygen transfer rates of 2.5 g O{sub 2} L{sup -1} h{sup -1} and 1.3 g O{sub 2} L{sup -1} h{sup -1} were recorded in the presence of Desmopan and silicone oil, respectively, which are in agreement with previously reported values in literature. These results suggest that mass transfer enhancements in TPPBs might correspond to an increase in a{sub g} rather than to the establishment of a high-performance gas/vector/water transfer pathway.

  15. 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

  16. 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.

  17. Oncolytic Adenoviruses in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    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.

  18. Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC.

    Science.gov (United States)

    Kim, Chong-Kook; Choi, Eun-Jeong; Choi, Sung-Hee; Park, Jeong-Sook; Haider, Khawaja Hasnain; Ahn, Woong Shick

    2003-08-01

    Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its

  19. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

    Science.gov (United States)

    Okamba, Faust René; Arella, Maximilien; Music, Nedzad; Jia, Jian Jun; Gottschalk, Marcelo; Gagnon, Carl A

    2010-07-05

    Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines. Copyright 2010 Elsevier Ltd. All rights reserved.

  20. Modulation transfer function (MTF) measurement method based on support vector machine (SVM)

    Science.gov (United States)

    Zhang, Zheng; Chen, Yueting; Feng, Huajun; Xu, Zhihai; Li, Qi

    2016-03-01

    An imaging system's spatial quality can be expressed by the system's modulation spread function (MTF) as a function of spatial frequency in terms of the linear response theory. Methods have been proposed to assess the MTF of an imaging system using point, slit or edge techniques. The edge method is widely used for the low requirement of targets. However, the traditional edge methods are limited by the edge angle. Besides, image noise will impair the measurement accuracy, making the measurement result unstable. In this paper, a novel measurement method based on the support vector machine (SVM) is proposed. Image patches with different edge angles and MTF levels are generated as the training set. Parameters related with MTF and image structure are extracted from the edge images. Trained with image parameters and the corresponding MTF, the SVM classifier can assess the MTF of any edge image. The result shows that the proposed method has an excellent performance on measuring accuracy and stability.

  1. Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

    Science.gov (United States)

    Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

    2007-07-01

    Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

  2. Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions.

    Science.gov (United States)

    Itaka, Keiji; Harada, Atsushi; Nakamura, Kozo; Kawaguchi, Hiroshi; Kataoka, Kazunori

    2002-01-01

    The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.

  3. Improvement of AD Biosynthesis Response to Enhanced Oxygen Transfer by Oxygen Vectors in Mycobacterium neoaurum TCCC 11979.

    Science.gov (United States)

    Su, Liqiu; Shen, Yanbing; Gao, Tian; Luo, Jianmei; Wang, Min

    2017-08-01

    In steroid biotransformation, soybean oil can improve the productivity of steroids by increasing substrate solubility and strengthen the cell membrane permeability. However, little is known of its role as oxygen carrier and its mechanism of promoting the steroid biotransformation. In this work, soybean oil used as oxygen vector for the enhancement of androst-4-ene-3,17-dione (AD) production by Mycobacterium neoaurum TCCC 11979 (MNR) was investigated. Upon the addition of 16% (v/v) soybean oil, the volumetric oxygen transfer coefficient (K L a) value increased by 44%, and the peak molar yield of AD (55.76%) was achieved. Analysis of intracellular cofactor levels showed high NAD + , ATP level, and a low NADH/NAD + ratio. Meanwhile, the two key enzymes of the tricarboxylic acid (TCA) cycle, namely, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, were upregulated after incubation with soybean oil. These enhancements induced by the increasing of oxygen supply showed positive effects on phytosterol (PS) bioconversion. Results could contribute to the understanding of effects of soybean oil as oxygen vector on steroid biotransformation and provided a convenient method for enhancing the efficiency of aerobic steroid biocatalysis.

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

    Science.gov (United States)

    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.

  5. Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Kasahara Noriyuki

    2010-05-01

    Full Text Available Abstract Background Gene transfer to the gastrointestinal (GI mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD, GI infections, and cancer. Methods We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G-pseudotyped lentiviral vectors (LV for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal or firefly-luciferase (LV-fLuc reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining. Results Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP. Conclusions We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

  6. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  7. Active and Passive 3D Vector Radiative Transfer with Preferentially-Aligned Ice Particles

    Science.gov (United States)

    Adams, I. S.; Munchak, S. J.; Pelissier, C.; Kuo, K. S.; Heymsfield, G. M.

    2017-12-01

    To support the observation of clouds and precipitation using combinations of radars and radiometers, a forward model capable of representing diverse sensing geometries for active and passive instruments is necessary for correctly interpreting and consistently combining multi-sensor measurements from ground-based, airborne, and spaceborne platforms. As such, the Atmospheric Radiative Transfer Simulator (ARTS) uses Monte Carlo integration to produce radar reflectivities and radiometric brightness temperatures for three-dimensional cloud and precipitation input fields. This radiative transfer framework is capable of efficiently sampling Gaussian antenna beams and fully accounting for multiple scattering. By relying on common ray-tracing tools, gaseous absorption models, and scattering properties, the model reproduces accurate and consistent radar and radiometer observables. While such a framework is an important component for simulating remote sensing observables, the key driver for self-consistent radiative transfer calculations of clouds and precipitation is scattering data. Research over the past decade has demonstrated that spheroidal models of frozen hydrometeors cannot accurately reproduce all necessary scattering properties at all desired frequencies. The discrete dipole approximation offers flexibility in calculating scattering for arbitrary particle geometries, but at great computational expense. When considering scattering for certain pristine ice particles, the Extended Boundary Condition Method, or T-Matrix, is much more computationally efficient; however, convergence for T-Matrix calculations fails at large size parameters and high aspect ratios. To address these deficiencies, we implemented the Invariant Imbedding T-Matrix Method (IITM). A brief overview of ARTS and IITM will be given, including details for handling preferentially-aligned hydrometeors. Examples highlighting the performance of the model for simulating space-based and airborne measurements

  8. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

    Science.gov (United States)

    Lehto, Taavi; Simonson, Oscar E; Mäger, Imre; Ezzat, Kariem; Sork, Helena; Copolovici, Dana-Maria; Viola, Joana R; Zaghloul, Eman M; Lundin, Per; Moreno, Pedro MD; Mäe, Maarja; Oskolkov, Nikita; Suhorutšenko, Julia; Smith, CI Edvard; Andaloussi, Samir EL

    2011-01-01

    Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice. PMID:21343913

  9. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Puntel, Mariana [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Ghulam, Muhammad A.K.M. [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Farrokhi, Catherine [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Kroeger, Kurt M.; Salem, Alireza [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Lacayo, Liliana [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Pechnick, Robert N. [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Psychiatry and Behavioral Neurosciences, David Geffen School of Medicine, University of California, Los Angeles, CA (United States); Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Palmer, Donna; Ng, Philip [Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 (United States); and others

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  10. A sensitive, support-vector-machine method for the detection of horizontal gene transfers in viral, archaeal and bacterial genomes.

    Science.gov (United States)

    Tsirigos, Aristotelis; Rigoutsos, Isidore

    2005-01-01

    In earlier work, we introduced and discussed a generalized computational framework for identifying horizontal transfers. This framework relied on a gene's nucleotide composition, obviated the need for knowledge of codon boundaries and database searches, and was shown to perform very well across a wide range of archaeal and bacterial genomes when compared with previously published approaches, such as Codon Adaptation Index and C + G content. Nonetheless, two considerations remained outstanding: we wanted to further increase the sensitivity of detecting horizontal transfers and also to be able to apply the method to increasingly smaller genomes. In the discussion that follows, we present such a method, Wn-SVM, and show that it exhibits a very significant improvement in sensitivity compared with earlier approaches. Wn-SVM uses a one-class support-vector machine and can learn using rather small training sets. This property makes Wn-SVM particularly suitable for studying small-size genomes, similar to those of viruses, as well as the typically larger archaeal and bacterial genomes. We show experimentally that the new method results in a superior performance across a wide range of organisms and that it improves even upon our own earlier method by an average of 10% across all examined genomes. As a small-genome case study, we analyze the genome of the human cytomegalovirus and demonstrate that Wn-SVM correctly identifies regions that are known to be conserved and prototypical of all beta-herpesvirinae, regions that are known to have been acquired horizontally from the human host and, finally, regions that had not up to now been suspected to be horizontally transferred. Atypical region predictions for many eukaryotic viruses, including the alpha-, beta- and gamma-herpesvirinae, and 123 archaeal and bacterial genomes, have been made available online at http://cbcsrv.watson.ibm.com/HGT_SVM/.

  11. The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro

    NARCIS (Netherlands)

    Grill, Jacques; Lamfers, Martine L. M.; van Beusechem, Victor W.; Dirven, Clemens M.; Pherai, D. Shareen; Kater, Mathijs; van der Valk, Paul; Vogels, Ronald; Vandertop, W. Peter; Pinedo, Herbert M.; Curiel, David T.; Gerritsen, Winald R.

    2002-01-01

    The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology

  12. 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...

  13. 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...

  14. Assessing the transferability of support vector machine model for estimation of global solar radiation from air temperature

    International Nuclear Information System (INIS)

    Chen, Ji-Long; Li, Guo-Sheng; Xiao, Bei-Bei; Wen, Zhao-Fei; Lv, Ming-Quan; Chen, Chun-Di; Jiang, Yi; Wang, Xiao-Xiao; Wu, Sheng-Jun

    2015-01-01

    Highlights: • Transferability of SVM in estimation of solar radiation is investigated. • Radiation at estimation site could be well estimated by SVM developed at source site. • A strategy for selecting a suitable source site is presented. • SVM accuracy is affected by distance and temperature difference between two sites. • RMSE of SVM shows logarithm or linearly relationship with altitude of source site. - Abstract: Exploring novel methods for estimation of global solar radiation from air temperature has been being a focus in many studies. This paper evaluates the transferability of support vector machines (SVM) for estimation of solar radiation in subtropical zone in China. Results suggest that solar radiation at one site (estimation site) could be well estimated by SVM model developed at another site (source site). The accuracy of estimation is affected by the distance and temperature difference between two sites, and altitude of source site. Higher correlations between RMSE of SVM and distance, and temperature differences are observed in northeastern region, increasing the reliability and confidence of SVM model developed at nearby stations. While lower correlations between RMSE and distance, and temperature differences are observed in southwest plateau region. When the altitude of estimation site is lower than 1200 m, RMSE show logarithm relationship with altitude of source sites where the altitude are lower than that of estimation site. Otherwise, RMSE show linearly relationship with altitude of source sites where the altitude are higher than 200 m but lower than that of the estimation site. This result suggests that solar radiation could be also estimated using SVM model developed at the site with similar but lower altitude. Based on these results, a strategy that takes into account the climatic conditions, topography, distance, and altitude for selecting a suitable source site is presented. The findings can guide and ease the appropriate choice of

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

    International Nuclear Information System (INIS)

    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.

  16. Metode Transfer Asam Nukleat sebagai Dasar Terapi Gen

    Directory of Open Access Journals (Sweden)

    Novi Silvia Hardiany

    2017-01-01

    Full Text Available Kemajuan ilmu biologi molekuler memberikan manfaat dalam bidang kedokteran untuk mengembangkanterapi gen. Tujuan terapi gen adalah untuk memperbaiki kerusakan gen atau mengganti gen yang rusakdengan gen yang normal. Pemindahan gen dilakukan dengan teknik transfeksi. Transfeksi merupakanproses pemindahan asam nukleat baik menggunakan vektor virus (transduksi atau menggunakan metodenonviral yaitu zat kimia, lipid dan metode fisik. Vektor virus yang digunakan pada transduksi adalahretrovirus, adenovirus, adeno-associated virus (AAV dan herpes simplex virus (HSV. Keberhasilantransfeksi ditentukan oleh berbagai faktor yang dapat dapat dinilai dengan menggunakan reporter sepertigreen fluorescence protein (GFP. Kata Kunci: terapi gen, transfeksi non viral, transduksi, vektor virus   Methods of Nucleic Acid Transfer as Basic Gene Therapy Abstract The advancement of molecular biology provides benefit in the field of medicine to develop genetherapy. The aim of gene therapy is to repair the genetic damage or to replace damaged gene with thenormal gene. Delivery of gene is carried out by transfection technique, a technique to transfer nucleic acidinto eukaryote cells either using viral vectors (known as transduction, and also using non viral methodsuch as chemical substance, lipid and physical method. Some of the viral vectors used in the transductionare retrovirus, adenovirus, Adeno-associated virus (AAV and Herpes Simplex Virus (HSV. The success oftransfection is determined by various factors which can be assessed using several reporters such as GreenFluorescence Protein (GFP. Key words: gene therapy, non viral transfection, transduction, viral vector. Normal 0 false false false IN X-NONE X-NONE

  17. Evaluation of modulation transfer function of optical lens system by support vector regression methodologies - A comparative study

    Science.gov (United States)

    Petković, Dalibor; Shamshirband, Shahaboddin; Saboohi, Hadi; Ang, Tan Fong; Anuar, Nor Badrul; Rahman, Zulkanain Abdul; Pavlović, Nenad T.

    2014-07-01

    The quantitative assessment of image quality is an important consideration in any type of imaging system. The modulation transfer function (MTF) is a graphical description of the sharpness and contrast of an imaging system or of its individual components. The MTF is also known and spatial frequency response. The MTF curve has different meanings according to the corresponding frequency. The MTF of an optical system specifies the contrast transmitted by the system as a function of image size, and is determined by the inherent optical properties of the system. In this study, the polynomial and radial basis function (RBF) are applied as the kernel function of Support Vector Regression (SVR) to estimate and predict estimate MTF value of the actual optical system according to experimental tests. Instead of minimizing the observed training error, SVR_poly and SVR_rbf attempt to minimize the generalization error bound so as to achieve generalized performance. The experimental results show that an improvement in predictive accuracy and capability of generalization can be achieved by the SVR_rbf approach in compare to SVR_poly soft computing methodology.

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

    Science.gov (United States)

    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.

  19. Simian virus 40 vectors for pulmonary gene therapy

    Directory of Open Access Journals (Sweden)

    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.

  20. Apparatus and method for transfer of information by means of a curl-free magnetic vector potential field

    International Nuclear Information System (INIS)

    Gelinas, R.C.

    1984-01-01

    A system for transmission of information using a curl-free magnetic vector potential radiation field. The system includes current-carrying apparatus for generating a magnetic vector potential field with a curl-free component coupled to apparatus for modulating the current applied to the field generating apparatus. Receiving apparatus includes a detector with observable properties that vary with the application of an applied curl-free magnetic vector potential field. Analyzing apparatus for determining the information content of modulation imposed on the curl-free vector potential field can be established in materials that are not capable of transmitting more common electromagnetic radiation

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

    Science.gov (United States)

    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.

  2. A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

    Science.gov (United States)

    Torrent, C; Gabus, C; Darlix, J L

    1994-02-01

    Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

  3. Investigation of Pear Drying Performance by Different Methods and Regression of Convective Heat Transfer Coefficient with Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Mehmet Das

    2018-01-01

    Full Text Available In this study, an air heated solar collector (AHSC dryer was designed to determine the drying characteristics of the pear. Flat pear slices of 10 mm thickness were used in the experiments. The pears were dried both in the AHSC dryer and under the sun. Panel glass temperature, panel floor temperature, panel inlet temperature, panel outlet temperature, drying cabinet inlet temperature, drying cabinet outlet temperature, drying cabinet temperature, drying cabinet moisture, solar radiation, pear internal temperature, air velocity and mass loss of pear were measured at 30 min intervals. Experiments were carried out during the periods of June 2017 in Elazig, Turkey. The experiments started at 8:00 a.m. and continued till 18:00. The experiments were continued until the weight changes in the pear slices stopped. Wet basis moisture content (MCw, dry basis moisture content (MCd, adjustable moisture ratio (MR, drying rate (DR, and convective heat transfer coefficient (hc were calculated with both in the AHSC dryer and the open sun drying experiment data. It was found that the values of hc in both drying systems with a range 12.4 and 20.8 W/m2 °C. Three different kernel models were used in the support vector machine (SVM regression to construct the predictive model of the calculated hc values for both systems. The mean absolute error (MAE, root mean squared error (RMSE, relative absolute error (RAE and root relative absolute error (RRAE analysis were performed to indicate the predictive model’s accuracy. As a result, the rate of drying of the pear was examined for both systems and it was observed that the pear had dried earlier in the AHSC drying system. A predictive model was obtained using the SVM regression for the calculated hc values for the pear in the AHSC drying system. The normalized polynomial kernel was determined as the best kernel model in SVM for estimating the hc values.

  4. A multi-layer discrete-ordinate method for vector radiative transfer in a vertically-inhomogeneous, emitting and scattering atmosphere. I - Theory. II - Application

    Science.gov (United States)

    Weng, Fuzhong

    1992-01-01

    A theory is developed for discretizing the vector integro-differential radiative transfer equation including both solar and thermal radiation. A complete solution and boundary equations are obtained using the discrete-ordinate method. An efficient numerical procedure is presented for calculating the phase matrix and achieving computational stability. With natural light used as a beam source, the Stokes parameters from the model proposed here are compared with the analytical solutions of Chandrasekhar (1960) for a Rayleigh scattering atmosphere. The model is then applied to microwave frequencies with a thermal source, and the brightness temperatures are compared with those from Stamnes'(1988) radiative transfer model.

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

    Science.gov (United States)

    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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  9. Deaths from Adenovirus in the US Military

    Centers for Disease Control (CDC) Podcasts

    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.

  10. Cross section measurement of vector meson quasi-photoproduction at high four-momentum transfer using the ZEUS detector at the HERA collider

    Energy Technology Data Exchange (ETDEWEB)

    Klimek, K.H.

    2001-11-01

    Photoproduction of vector mesons, {gamma}p {yields} VY, where Y is the system into which the proton dissociates, has been measured in e{sup +}p interactions with the ZEUS detector at HERA using an integrated luminosity of 24 pb{sup -1}. The data were taken at a photon-proton center-of-mass energy W {approx_equal} 100 GeV and extend up to -t = 10 GeV{sup 2}, where t is the square of the four-momentum transferred to the vector meson. The differential cross section d{sigma}/dt for {rho}{sup 0}, {phi} and J/{psi} production are compared to the expectations of phenomenological models. (orig.)

  11. Investigating the impact of the gluon saturation effects on the momentum transfer distributions for the exclusive vector meson photoproduction in hadronic collisions

    Directory of Open Access Journals (Sweden)

    V.P. Gonçalves

    2017-05-01

    Full Text Available The exclusive vector meson production cross section is one of the most promising observables to probe the high energy regime of the QCD dynamics. In particular, the squared momentum transfer (t distributions are an important source of information about the spatial distribution of the gluons in the hadron and about fluctuations of the color fields. In this paper we complement previous studies on exclusive vector meson photoproduction in hadronic collisions presenting a comprehensive analysis of the t-spectrum measured in exclusive ρ, ϕ and J/Ψ photoproduction in pp and PbPb collisions at the LHC. We compute the differential cross sections taking into account gluon saturation effects and compare the predictions with those obtained in the linear regime of the QCD dynamics. Our results show that gluon saturation suppresses the magnitude of the cross sections and shifts the position of the dips towards smaller values of t.

  12. 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 ...

  13. Ratio of spin transfer parameters dt/rt in d(p vector, n vector)pp quasi-elastic scattering

    International Nuclear Information System (INIS)

    Abegg, R.; Green, W.; Greeniaus, L.G.; Miller, C.A.; Bardyopadhyay, D.; Birchall, J.; Davis, C.A.; Davison, N.E.; Page, S.A.; Ramsay, W.D.; van Oers, W.T.H.; Lapointe, C.; Moss, G.A.; Tkachuk, R.R.

    1988-05-01

    The ratio of spin transfer parameters d t /r t for the quasi-elastic process d(p,n)pp has been measured at four energies between 200 and 500 MeV at a neutron scattering angle of 9 degrees. From this, the following values of D t /R t for free np scattering have been deduced: -0.0190 ± 0.0072 (T p = 223 MeV); -0.2328 ± 0.0057 (324 MeV); -0.3731 ± 0.0068 (425 MeV); -0.4892 ± 0.0107 (492 MeV). These values have a noticeable effect on present day phase shift solutions. The magnitude of the ε 1 mixing parameter is reduced and other phase shifts are smoother around 300 MeV. (Author) (17 refs., 2 tabs., 3 figs.)

  14. 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.

  15. Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

    Science.gov (United States)

    Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

    2017-01-01

    A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

  16. Enfermedad neurologica por adenovirus Neurologic disease due to adenovirus infection

    Directory of Open Access Journals (Sweden)

    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

  17. A Drosophila melanogaster hobo-white + vector mediates low frequency gene transfer in D. vlrllls with full Interspecific white + complementation

    Science.gov (United States)

    Transformation of a Drosophila virilis white mutant host strain was attempted by using a hobo vector containing the D. melanogaster mini-white+ cassette (H[w+, hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (...

  18. 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  1. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

    Science.gov (United States)

    Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

    1999-01-01

    Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

  2. Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells).

    Science.gov (United States)

    Howarth, Joanna L; Lee, Youn Bok; Uney, James B

    2010-02-01

    In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.

  3. Components of Adenovirus Genome Packaging

    Science.gov (United States)

    Ahi, Yadvinder S.; Mittal, Suresh K.

    2016-01-01

    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

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

    Science.gov (United States)

    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.

  5. Improved results for the 2H(d, n)3He transverse vector polarization-transfer coefficient Kyy'(0o) at low energies

    International Nuclear Information System (INIS)

    Roper, C.D.; Dunham, J.D.; Clegg, T.B.; Mendez, A.J.; Tornow, W.; Walter, R.L.

    2010-01-01

    Measurements of the 2 H(d, n) 3 He transverse vector polarization-transfer coefficient K y y' at 0 o . are reported for 29 outgoing neutron energies between 3.94 and 8.47 MeV. Our new results determine K y y' (0 o ) more accurately than previous data, especially for neutron energies below 5 MeV. Low-energy data for this reaction are important both as a high-intensity source of highly polarized neutrons for nuclear physics studies with polarized neutron beams, and as a test of the emerging theoretical descriptions of the four-body system, where recently substantial progress has been made. (author)

  6. Improved Results for the H-2(d, n)(3) He Transverse Vector Polarization- Transfer Coefficient K-y(y)' (0 degrees) at Low Energies

    International Nuclear Information System (INIS)

    Roper, C.D.; Clegg, T.; Dunham, J.D.; Mendez, Anthony J. II; Tornow, W.; Walter, R.L.

    2010-01-01

    Measurements of the H-2(d, n)(3) He transverse vector polarization-transfer coefficient K-y(y)' at 0 degrees. are reported for 29 outgoing neutron energies between 3.94 and 8.47MeV. Our new results determine K-y(y)' (0 degrees) more accurately than previous data, especially for neutron energies below 5MeV. Low-energy data for this reaction are important both as a high-intensity source of highly polarized neutrons for nuclear physics studies with polarized neutron beams, and as a test of the emerging theoretical descriptions of the four-body system, where recently substantial progress has been made.

  7. 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.

  8. Transfer

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne; Aarkrog, Vibe

    Bogen er den første samlede indføring i transfer på dansk. Transfer kan anvendes som praksis-filosofikum. Den giver en systematisk indsigt til den studerende, der spørger: Hvordan kan teoretisk viden bruges til at reflektere over handlinger i situationer, der passer til min fremtidige arbejdsplads?...

  9. 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.

  10. 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.

  11. 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...

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

    Science.gov (United States)

    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

  13. 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

  14. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    Science.gov (United States)

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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

  17. 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.

  18. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  2. TRANSFER

    African Journals Online (AJOL)

    This paper reports on further studies on long range energy transfer between curcumine as donor and another thiazine dye, thionine, which is closely related to methylene blue as energy harvester (Figure 1). Since thionine is known to have a higher quantum yield of singlet oxygen sensitization than methylene blue [8], it is ...

  3. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    Energy Technology Data Exchange (ETDEWEB)

    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).

  4. Reed beds may facilitate transfer of tributyltin from aquatic to terrestrial ecosystems through insect vectors in the Archipelago Sea, SW Finland.

    Science.gov (United States)

    Lilley, Thomas M; Meierjohann, Axel; Ruokolainen, Lasse; Peltonen, Jani; Vesterinen, Eero; Kronberg, Leif; Nikinmaa, Mikko

    2012-08-01

    Due to their adsorptive behavior, organotin compounds (OTCs), such as tributyltin (TBT), are accumulated in aquatic sediments. They resist biodegradation and, despite a ban in 2008, are a potential source for future exposure. Sediment OTCs have mostly been measured from sites of known high concentrations such as ports, shipping lanes, and marine dredging waste sites. The possible flow of OTCs from marine to terrestrial ecosystems, however, has not been studied. In the present study, the authors assessed whether sediments in common reed beds (Phragmites australis) accumulate TBT and whether chironomid (Diptera: Chironomidae) communities developing in reed-bed sediments act as vectors in the transfer of TBT from aquatic to terrestrial ecosystems in the Airisto channel, Archipelago Sea. The authors also investigated whether distance from the only known source and depth and TBT concentration of the adjacent shipping lane affect reed-bed concentrations. Thirty-six sites along the Airisto channel were sampled at 2-km intervals with triplicate samples from reed beds and the adjacent shipping lane for sediment and seven reed-bed sites for chironomids, and these were analyzed with an solid phase extraction liquid chromatography tamdem mass spectrometry method. The closer to the source the sample site was, the higher the measured TBT concentrations were; and the deeper the shipping lane, the lower the concentration of TBT in reed-bed sediments. The chironomid TBT concentrations correlated with reed-bed sediment TBT concentrations and showed evidence of accumulation. Therefore, TBT may be transferred, through the food web, from aquatic to terrestrial ecosystems relatively close to a source through ecosystem boundaries, such as common reed beds, which are areas of high insect biomass production in the Archipelago Sea. Copyright © 2012 SETAC.

  5. Precise determination of the spin-transfer coefficient K sub N sub sub N sub ' for n-vector p elastic scattering at 187 MeV

    CERN Document Server

    Wissink, S W; Franklin, W A; Jacobs, W W; Peterson, T; Rinckel, T; Sowinski, J; Stephenson, E J; Wolanski, M; Yang, H

    2000-01-01

    We have measured the normal-component spin-transfer observable K sub N sub sub N sub ' for n-vector p elastic scattering to a precision of approx +-0.014 at 12 angles in the far-backward region (110 deg. <= theta sub c sub m <= 170 deg. ) at a neutron bombarding energy of 187 MeV. This kinematic regime is of particular interest in that the scattering is mediated predominantly through exchange of a single charged pion, and hence is expected to depend sensitively on the strength of the pi sup+-N N coupling constant g sup 2 sub c , a quantity whose precise value remains highly controversial. The measurements described here were carried out at the upgraded Polarized Neutron Facility at IUCF. Details of our experimental apparatus, and the analysis procedures we employed, will be discussed, paying particular attention to potential sources of systematic error and their effect on the data. Our primary result is that preliminary values for K sub N sub sub N sub ' appear to agree most closely with the predictions...

  6. Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

    Directory of Open Access Journals (Sweden)

    Ashley T Martino

    2009-08-01

    Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

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

    Science.gov (United States)

    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.

  8. Gene transfer therapy in vascular diseases.

    Science.gov (United States)

    McKay, M J; Gaballa, M A

    2001-01-01

    Somatic gene therapy of vascular diseases is a promising new field in modern medicine. Recent advancements in gene transfer technology have greatly evolved our understanding of the pathophysiologic role of candidate disease genes. With this knowledge, the expression of selective gene products provides the means to test the therapeutic use of gene therapy in a multitude of medical conditions. In addition, with the completion of genome sequencing programs, gene transfer can be used also to study the biologic function of novel genes in vivo. Novel genes are delivered to targeted tissue via several different vehicles. These vectors include adenoviruses, retroviruses, plasmids, plasmid/liposomes, and oligonucleotides. However, each one of these vectors has inherent limitations. Further investigations into developing delivery systems that not only allow for efficient, targeted gene transfer, but also are stable and nonimmunogenic, will optimize the clinical application of gene therapy in vascular diseases. This review further discusses the available mode of gene delivery and examines six major areas in vascular gene therapy, namely prevention of restenosis, thrombosis, hypertension, atherosclerosis, peripheral vascular disease in congestive heart failure, and ischemia. Although we highlight some of the recent advances in the use of gene therapy in treating vascular disease discovered primarily during the past two years, many excellent studies published during that period are not included in this review due to space limitations. The following is a selective review of practical uses of gene transfer therapy in vascular diseases. This review primarily covers work performed in the last 2 years. For earlier work, the reader may refer to several excellent review articles. For instance, Belalcazer et al. (6) reviewed general aspects of somatic gene therapy and the different vehicles used for the delivery of therapeutic genes. Gene therapy in restenosis and stimulation of

  9. Vector analysis

    CERN Document Server

    Newell, Homer E

    2006-01-01

    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  10. About vectors

    CERN Document Server

    Hoffmann, Banesh

    1975-01-01

    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

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

    Science.gov (United States)

    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.

  12. Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function

    NARCIS (Netherlands)

    Blits, B; Oudega, M.; Boer, G J; Bartlett Bunge, M; Verhaagen, J

    2003-01-01

    To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding

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

    Science.gov (United States)

    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.

  14. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ

    1978-01-01

    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  15. 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...

  16. 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

  17. 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

  18. The structural basis for the integrity of adenovirus Ad3 dodecahedron.

    Directory of Open Access Journals (Sweden)

    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.

  19. Diseño y construcción de vectores de transferencia para la obtención de virus vaccinia Ankara modificado (MVA recombinantes Design and construction of transfer vectors in order to obtain recombinant modified vaccinia virus Ankara (MVA

    Directory of Open Access Journals (Sweden)

    M. F. Ferrer

    2007-09-01

    Full Text Available El virus vaccinia Ankara modificado (MVA constituye un buen candidato para el desarrollo de vectores virales de expresión no replicativos porque no replica en la mayoría de las células de mamíferos. Para la producción de MVA recombinantes es fundamental disponer de vectores de transferencia que, por recombinación homóloga con el genoma viral, permitan introducir los genes de interés en regiones no esenciales para la replicación in vitro. En este trabajo se diseñaron y obtuvieron los vectores de transferencia denominados VT-MHA y VT-MTK que portan las regiones correspondientes a las posiciones 1-303 y 608-948 del gen MVA165R y 1-244 y 325-534 del gen MVA086R, respectivamente, las que flanquean un sitio de clonado múltiple para la inserción de los genes foráneos. En dichos vectores se clonaron los casetes para la expresión de los genes lac Z o uid A, y la actividad de las enzimas marcadoras b-galactosidasa y b-glucuronidasa se confirmó in situ. Además, utilizando el vector denominado VT-MTK-GUS, se obtuvieron y aislaron MVA recombinantes puros que portan y expresan el gen uid A. Los resultados obtenidos constituyen las herramientas básicas para establecer la metodología de obtención de MVA recombinantes, con el propósito de desarrollar localmente vectores virales no replicativos candidatos a vacunas.Modified Vaccinia virus Ankara (MVA constitutes a good candidate for the development of non-replicative expression viral vectors because it does not replicate in most of mammalian cells. It is essential, for the production of recombinant MVA, the availability of transfer vectors which allow the introduction of desired genes into non-essential regions for in vitro viral replication, by homologous recombination with the viral genome. In the present work, the transfer vectors named VT-MHA and VT-MTK were designed and obtained. They carried genomic regions corresponding to 1- 303 and 608-948 positions of the MVA165R gene and 1-244 and

  20. Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

    International Nuclear Information System (INIS)

    Rivera, Angel A.; Wang Minghui; Suzuki, Kaori; Uil, Taco G.; Krasnykh, Victor; Curiel, David T.; Nettelbeck, Dirk M.

    2004-01-01

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

  1. Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

    Science.gov (United States)

    Sena-Esteves, M; Camp, S M; Alroy, J; Breakefield, X O; Kaye, E M

    2000-03-20

    Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

  2. 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

  3. 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.

  4. Human adenovirus-36 and childhood obesity.

    Science.gov (United States)

    Atkinson, Richard L

    2011-09-01

    There is increasing evidence that obesity in humans is associated with infection with human adenovirus-36 (Adv36). Infection of experimental animals with Adv36 demonstrates that this virus causes obesity. Human studies have shown a prevalence of Adv36 infection of 30% or greater in obese adult humans, but a correlation with obesity has not always been demonstrated. In contrast, three published studies and one presented study with a total of 559 children all show that there is an increase in prevalence of Adv36 infection in obese children (28%) compared to non-obese children (10%). The explanation for the apparently more robust correlation of Adv36 infection with obesity in children vs. adults is not clear. The data in animals and people suggests that Adv36 has contributed to the worldwide increase in childhood obesity. More research is needed to identify prevalences and consequences of Adv36 infection in people of all age groups and geographic locations.

  5. Adenovirus 36 DNA in human adipose tissue.

    Science.gov (United States)

    Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

    2015-12-01

    Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

  6. Vector analysis

    CERN Document Server

    Brand, Louis

    2006-01-01

    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  7. 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,

  8. Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus

    Directory of Open Access Journals (Sweden)

    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.

  9. Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

    Directory of Open Access Journals (Sweden)

    Stefania Piersanti

    Full Text Available Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD canine adenovirus type 2 vectors (CAV-2 are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

  10. 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.

  11. 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

  12. Measurement of guided mode wave vectors by analysis of the transfer matrix obtained with multi-emitters and multi-receivers in contact

    Energy Technology Data Exchange (ETDEWEB)

    Minonzio, Jean-Gabriel; Talmant, Maryline; Laugier, Pascal, E-mail: jean-gabriel.minonzio@upmc.fr [UPMC Univ Paris 06, UMR 7623, LIP, 15 rue de l' ecole de medecine F-75005, Paris (France)

    2011-01-01

    Different quantitative ultrasound techniques are currently developed for clinical assessment of human bone status. This paper is dedicated to axial transmission: emitters and receivers are linearly arranged on the same side of the skeletal site, preferentially the forearm. In several clinical studies, the signal velocity of the earliest temporal event has been shown to discriminate osteoporotic patients from healthy subjects. However, a multi parameter approach might be relevant to improve bone diagnosis and this be could be achieved by accurate measurement of guided waves wave vectors. For clinical purposes and easy access to the measurement site, the length probe is limited to about 10 mm. The limited number of acquisition scan points on such a short distance reduces the efficiency of conventional signal processing techniques, such as spatio-temporal Fourier transform. The performance of time-frequency techniques was shown to be moderate in other studies. Thus, optimised signal processing is a critical point for a reliable estimate of guided mode wave vectors. Toward this end, a technique, taking benefit of using both multiple emitters and multiple receivers, is proposed. The guided mode wave vectors are obtained using a projection in the singular vectors basis. Those are determined by the singular values decomposition of the transmission matrix between the two arrays at different frequencies. This technique enables us to recover accurately guided waves wave vectors for moderately large array.

  13. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...

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

    Science.gov (United States)

    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.

  15. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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...

  1. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  2. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

    Science.gov (United States)

    Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

    2013-01-01

    Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (Pgenetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

  5. Equivalent Vectors

    Science.gov (United States)

    Levine, Robert

    2004-01-01

    The cross-product is a mathematical operation that is performed between two 3-dimensional vectors. The result is a vector that is orthogonal or perpendicular to both of them. Learning about this for the first time while taking Calculus-III, the class was taught that if AxB = AxC, it does not necessarily follow that B = C. This seemed baffling. The…

  6. Default assembly of early adenovirus chromatin

    International Nuclear Information System (INIS)

    Spector, David J.

    2007-01-01

    In adenovirus particles, the viral nucleoprotein is organized into a highly compacted core structure. Upon delivery to the nucleus, the viral nucleoprotein is very likely to be remodeled to a form accessible to the transcription and replication machinery. Viral protein VII binds to intra-nuclear viral DNA, as do at least two cellular proteins, SET/TAF-Iβ and pp32, components of a chromatin assembly complex that is implicated in template remodeling. We showed previously that viral DNA-protein complexes released from infecting particles were sensitive to shearing after cross-linking with formaldehyde, presumably after transport of the genome into the nucleus. We report here the application of equilibrium-density gradient centrifugation to the analysis of the fate of these complexes. Most of the incoming protein VII was recovered in a form that was not cross-linked to viral DNA. This release of protein VII, as well as the binding of SET/TAF-Iβ and cellular transcription factors to the viral chromatin, did not require de novo viral gene expression. The distinct density profiles of viral DNA complexes containing protein VII, compared to those containing SET/TAF-Iβ or transcription factors, were consistent with the notion that the assembly of early viral chromatin requires both the association of SET/TAF-1β and the release of protein VII

  7. Latest Insights on Adenovirus Structure and Assembly

    Directory of Open Access Journals (Sweden)

    Carmen San Martín

    2012-05-01

    Full Text Available Adenovirus (AdV capsid organization is considerably complex, not only because of its large size (~950 Å and triangulation number (pseudo T = 25, but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  8. 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.

  9. 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.

  10. Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers

    DEFF Research Database (Denmark)

    Hansen, A C; Grunwald, T; Lund, Anders Henrik

    2001-01-01

    could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold...

  11. 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.

  12. Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy

    Directory of Open Access Journals (Sweden)

    Siegal Gene P

    2007-10-01

    Full Text Available Abstract Background Human adenovirus serotype 5 (Ad5 has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR, and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery. Results In this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTDtat was mediated by binding of the positively charged PTDtat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTDtat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTDtat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique. Conclusion Genetic incorporation of the PTDtat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTDtat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy

  13. 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.

  14. Adenovirus respiratory tract infections in Peru.

    Directory of Open Access Journals (Sweden)

    Julia S Ampuero

    Full Text Available BACKGROUND: Currently, there is a paucity of data regarding human adenovirus (HAdv circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. METHODS/PRINCIPAL FINDINGS: Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI or severe acute respiratory infection (SARI were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. CONCLUSIONS/SIGNIFICANCE: HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness.

  15. Adenovirus respiratory tract infections in Peru.

    Science.gov (United States)

    Ampuero, Julia S; Ocaña, Víctor; Gómez, Jorge; Gamero, María E; Garcia, Josefina; Halsey, Eric S; Laguna-Torres, V Alberto

    2012-01-01

    Currently, there is a paucity of data regarding human adenovirus (HAdv) circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI) or severe acute respiratory infection (SARI) were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness.

  16. Adenovirus Respiratory Tract Infections in Peru

    Science.gov (United States)

    Ampuero, Julia S.; Ocaña, Víctor; Gómez, Jorge; Gamero, María E.; Garcia, Josefina; Halsey, Eric S.; Laguna-Torres, V. Alberto

    2012-01-01

    Background Currently, there is a paucity of data regarding human adenovirus (HAdv) circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. Methods/Principal Findings Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI) or severe acute respiratory infection (SARI) were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. Conclusions/Significance HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness. PMID:23056519

  17. Genomic stability of adipogenic human adenovirus 36.

    Science.gov (United States)

    Nam, J-H; Na, H-N; Atkinson, R L; Dhurandhar, N V

    2014-02-01

    Human adenovirus Ad36 increases adiposity in several animal models, including rodents and non-human primates. Importantly, Ad36 is associated with human obesity, which has prompted research to understand its epidemiology and to develop a vaccine to prevent a subgroup of obesity. For this purpose, understanding the genomic stability of Ad36 in vivo and in vitro infections is critical. Here, we examined whether in vitro cell passaging over a 14-year period introduced any genetic variation in Ad36. We sequenced the whole genome of Ad36-which was plaque purified in 1998 from the original strain obtained from American Type Culture Collection, and passaged approximately 12 times over the past 14 years (Ad36-2012). This DNA sequence was compared with a previously published sequence of Ad36 likely obtained from the same source (Ad36-1988). Compared with Ad36-1988, only two nucleotides were altered in Ad36-2012: a T insertion at nucleotide 1862, which may induce early termination of the E1B viral protein, and a T➝C transition at nucleotide 26 136. Virus with the T insertion (designated Ad36-2012-T6) was mixed with wild-type virus lacking the T insertion (designated Ad36-2012-T5) in the viral stock. The transition at nucleotide 26 136 does not change the encoded amino acid (aspartic acid) in the pVIII viral protein. The rate of genetic variation in Ad36 is ∼2.37 × 10(-6) mutations/nucleotide/passage. Of particular importance, there were no mutations in the E4orf1 gene, the critical gene for producing obesity. This very-low-variation rate should reduce concerns about genetic variability when developing Ad36 vaccines or developing assays for detecting Ad36 infection in populations.

  18. 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...

  19. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B

    2011-01-01

    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom

  20. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    Science.gov (United States)

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  1. 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

  2. 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.

  3. VECTOR INTEGRATION

    NARCIS (Netherlands)

    Thomas, E. G. F.

    2012-01-01

    This paper deals with the theory of integration of scalar functions with respect to a measure with values in a, not necessarily locally convex, topological vector space. It focuses on the extension of such integrals from bounded measurable functions to the class of integrable functions, proving

  4. 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, ...

  5. 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.

  6. Bioaccumulation of animal adenoviruses in the pink shrimp

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. 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.

  10. An introduction to vectors, vector operators and vector analysis

    CERN Document Server

    Joag, Pramod S

    2016-01-01

    Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

  11. 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.

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

    Science.gov (United States)

    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.

  13. Measurement of K/sub NN/, K/sub LL/ in p vector d → n vector X and p vector 9Be → n vector X at 800 MeV

    International Nuclear Information System (INIS)

    Riley, P.J.; Hollas, C.L.; Newsom, C.R.

    1980-01-01

    The spin transfer parameters, K/sub NN/ and K/sub LL/, have been measured in p vector d → n vector X and p vector 9 Be → n vector X at 0 0 and 800 MeV. The rather large values of K/sub LL/ demonstrate that this transfer mechanism will provide a useful source of polarized neutrons at LAMPF energies

  14. 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

  15. Scattering and emission from inhomogeneous vegetation canopy and alien target beneath by using three-dimensional vector radiative transfer (3D-VRT) equation

    International Nuclear Information System (INIS)

    Jin Yaqiu; Liang Zichang

    2005-01-01

    To solve the 3D-VRT equation for the model of spatially inhomogeneous scatter media, the finite enclosure of the scatter media is geometrically divided, in both vertical z and transversal (x,y) directions, to form very thin multi-boxes. The zeroth order emission, first-order Mueller matrix of each thin box and an iterative approach of high-order radiative transfer are applied to derive high-order scattering and emission of whole inhomogeneous scatter media. Numerical results of polarized brightness temperature at microwave frequency and under different radiometer resolutions from inhomogeneous scatter model such as vegetation canopy and alien target beneath canopy are simulated and discussed

  16. Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice - role of regulatory T cells.

    Science.gov (United States)

    Singpiel, Alena; Kramer, Julia; Maus, Regina; Stolper, Jennifer; Bittersohl, Lara Friederike; Gauldie, Jack; Kolb, Martin; Welte, Tobias; Sparwasser, Tim; Maus, Ulrich A

    2018-03-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγ pos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  18. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  19. 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.

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

    Science.gov (United States)

    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

  1. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    . Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

  2. 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.

  3. 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

  4. 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

  5. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.)

  8. 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...

  9. 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Concentration of Reovirus and Adenovirus from Sewage and Effluents by Protamine Sulfate (Salmine) Treatment 1

    Science.gov (United States)

    England, Beatrice

    1972-01-01

    Protamine sulfate was employed to recover reoviruses, adenoviruses, and certain enteroviruses from sewage and treated effluents; 50- to 400-fold concentration of viral content was achieved. PMID:4342842

  13. 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...

  14. 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.

  15. 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

  16. Development of a replication defective adenovirus 5 vector expressing porcine interleukin-18 and a mutated analog

    Science.gov (United States)

    Cell-mediated immune responses against swine pathogens are sometimes necessary to elicit durable protective immunity. Cell mediated or Th1 immunity is dependent on the coordinated expression of several cytokines, including interferon-gamma to assist in the production of antigen-specific cytotoxic T...

  17. The process of lipid storage in insect oocytes: The involvement of β-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas' disease vector Panstrongylus megistus (Hemiptera: Reduviidae).

    Science.gov (United States)

    Fruttero, Leonardo L; Leyria, Jimena; Ramos, Fabián O; Stariolo, Raúl; Settembrini, Beatriz P; Canavoso, Lilián E

    2017-01-01

    Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus

  18. Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

    Science.gov (United States)

    Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

    2010-06-22

    Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

  19. Oncolytic adenovirus Ad657 for systemic virotherapy against prostate cancer

    Directory of Open Access Journals (Sweden)

    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

  20. Effects of infection with recombinant adenovirus on human vascular endothelial and smooth muscle cells

    NARCIS (Netherlands)

    Quax, P.H.A.; Lamfers, M.L.M.; Grimbergen, J.M.; Teeling, J.; Hoeben, R.C.; Nieuw Amerongen, G.P. van; Hinsbergh, V.W.M. van

    1996-01-01

    The plasminogen activation (PA) system is involved in vascular remodelling. Modulating its activity in vascular cells might be a way to interfere in processes such as angiogenesis and restenosis. Adenoviral vectors have become a favourable tool for direct gene transfer into vascular cells. In the

  1. Characterizations of the random order values by Harsanyi payoff vectors

    NARCIS (Netherlands)

    Derks, J.; van der Laan, G.; Vasil'ev, V.

    2006-01-01

    A Harsanyi payoff vector (see Vasil'ev in Optimizacija Vyp 21:30-35, 1978) of a cooperative game with transferable utilities is obtained by some distribution of the Harsanyi dividends of all coalitions among its members. Examples of Harsanyi payoff vectors are the marginal contribution vectors. The

  2. 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.

  3. 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.

  4. Exporting vector muscles for facial animation

    NARCIS (Netherlands)

    Bui, T.D.; Butz, Andreas; Kruger, Antonio; Heylen, Dirk K.J.; Olivier, Patrick; Nijholt, Antinus; Poel, Mannes

    2003-01-01

    In this paper we introduce a method of exporting vector muscles from one 3D face to another for facial animation. Starting from a 3D face with an extended version of Waters’ linear muscle system, we transfer the linear muscles to a target 3D face.We also transfer the region division, which is used

  5. Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. 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

  8. 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)

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Canine adenovirus type 1 in a fennec fox (Vulpes zerda).

    Science.gov (United States)

    Choi, Jeong-Won; Lee, Hyun-Kyoung; Kim, Seong-Hee; Kim, Yeon-Hee; Lee, Kyoung-Ki; Lee, Myoung-Heon; Oem, Jae-Ku

    2014-12-01

    A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing.

  12. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla

    Directory of Open Access Journals (Sweden)

    Ludwig Carsten

    2010-11-01

    Full Text Available Abstract Adenoviruses (AdV broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL, preterminal protein (pTP and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2 - 10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B. Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

  13. Adenovirus chromatin structure at different stages of infection

    Energy Technology Data Exchange (ETDEWEB)

    Daniell, E.; Groff, D.E.; Fedor, M.J.

    1981-12-01

    The authors investigated the structure of adenovirus deoxyribonecleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parental DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with (/sup 3/)thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure they identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. The authors found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

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

    Science.gov (United States)

    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.

  15. 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

  16. Ex vivo adenoviral vector-mediated neurotrophin gene transfer to olfactory ensheathing glia : effects on rubrospinal tract regeneration, lesion size, and functional recovery after implantation in the injured rat spinal cord

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Plant, Giles W; Hamers, Frank P T; Wortel, Joke; Blits, Bas; Dijkhuizen, Paul A; Gispen, Willem Hendrik; Boer, Gerard J; Verhaagen, J.

    2003-01-01

    The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived

  17. Raster images vectorization system

    OpenAIRE

    Genytė, Jurgita

    2006-01-01

    The problem of raster images vectorization was analyzed and researched in this work. Existing vectorization systems are quite expensive, the results are inaccurate, and the manual vectorization of a large number of drafts is impossible. That‘s why our goal was to design and develop a new raster images vectorization system using our suggested automatic vectorization algorithm and the way to record results in a new universal vectorial file format. The work consists of these main parts: analysis...

  18. Kochen-Specker vectors

    International Nuclear Information System (INIS)

    Pavicic, Mladen; Merlet, Jean-Pierre; McKay, Brendan; Megill, Norman D

    2005-01-01

    We give a constructive and exhaustive definition of Kochen-Specker (KS) vectors in a Hilbert space of any dimension as well as of all the remaining vectors of the space. KS vectors are elements of any set of orthonormal states, i.e., vectors in an n-dimensional Hilbert space, H n , n≥3, to which it is impossible to assign 1s and 0s in such a way that no two mutually orthogonal vectors from the set are both assigned 1 and that not all mutually orthogonal vectors are assigned 0. Our constructive definition of such KS vectors is based on algorithms that generate MMP diagrams corresponding to blocks of orthogonal vectors in R n , on algorithms that single out those diagrams on which algebraic (0)-(1) states cannot be defined, and on algorithms that solve nonlinear equations describing the orthogonalities of the vectors by means of statistically polynomially complex interval analysis and self-teaching programs. The algorithms are limited neither by the number of dimensions nor by the number of vectors. To demonstrate the power of the algorithms, all four-dimensional KS vector systems containing up to 24 vectors were generated and described, all three-dimensional vector systems containing up to 30 vectors were scanned, and several general properties of KS vectors were found

  19. 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

  20. 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.

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

    Science.gov (United States)

    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.

  2. 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

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

    Science.gov (United States)

    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.

  4. 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.

  5. Vector regression introduced

    Directory of Open Access Journals (Sweden)

    Mok Tik

    2014-06-01

    Full Text Available This study formulates regression of vector data that will enable statistical analysis of various geodetic phenomena such as, polar motion, ocean currents, typhoon/hurricane tracking, crustal deformations, and precursory earthquake signals. The observed vector variable of an event (dependent vector variable is expressed as a function of a number of hypothesized phenomena realized also as vector variables (independent vector variables and/or scalar variables that are likely to impact the dependent vector variable. The proposed representation has the unique property of solving the coefficients of independent vector variables (explanatory variables also as vectors, hence it supersedes multivariate multiple regression models, in which the unknown coefficients are scalar quantities. For the solution, complex numbers are used to rep- resent vector information, and the method of least squares is deployed to estimate the vector model parameters after transforming the complex vector regression model into a real vector regression model through isomorphism. Various operational statistics for testing the predictive significance of the estimated vector parameter coefficients are also derived. A simple numerical example demonstrates the use of the proposed vector regression analysis in modeling typhoon paths.

  6. PCR Analysis of Egyptian Respiratory Adenovirus Isolates, Including Identification of Species, Serotypes, and Coinfections

    National Research Council Canada - National Science Library

    Metzgar, David; Osuna, Miguel; Yingst, Samuel; Rakha, Magda; Earhart, Kenneth; Elyan, Diaa; Esmat, Hala; Saad, Magdi D; Kajon, Adriana; Wu, Jianguo; Gray, Gregory C; Ryan, Margaret A; Russell, Kevin L

    2005-01-01

    Eighty-eight adenovirus (Ad) isolates and associated clinical data were collected from walk-in patients with influenza-like illness in Egypt during routine influenza surveillance from 1999 through 2002...

  7. 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.

  8. New adenoviruses from new primate hosts - growing diversity reveals taxonomic weak points

    Czech Academy of Sciences Publication Activity Database

    Dadáková, E.; Chrudimský, Tomáš; Brožová, K.; Modrý, David; Celer, V.; Hrazdilová, K.

    2017-01-01

    Roč. 107, February (2017), s. 305-307 ISSN 1055-7903 Institutional support: RVO:60077344 Keywords : adenovirus * primate * phylogeny * taxonomy Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.419, year: 2016

  9. Detection of enteric Adenoviruses in South-African waters using gene probes

    CSIR Research Space (South Africa)

    Genthe, Bettina

    1995-01-01

    Full Text Available Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultra filtration and analysed...

  10. 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

  11. Partial characterization of new adenoviruses found in lizards.

    Science.gov (United States)

    Ball, Inna; Behncke, Helge; Schmidt, Volker; Geflügel, F T A; Papp, Tibor; Stöhr, Anke C; Marschang, Rachel E

    2014-06-01

    In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species.

  12. Development of an immunotherapeutic adenovirus targeting hormone-independent prostate cancer

    Directory of Open Access Journals (Sweden)

    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

  13. 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.

  14. 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 ...

  15. 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...

  16. 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 ...

  17. VectorBase

    Data.gov (United States)

    U.S. Department of Health & Human Services — VectorBase is a Bioinformatics Resource Center for invertebrate vectors. It is one of four Bioinformatics Resource Centers funded by NIAID to provide web-based...

  18. Travel and disease vector ticks.

    Science.gov (United States)

    McGarry, John W

    2011-03-01

    There are approximately twenty species of hard (ixodid) ticks worldwide that frequently affect human populations, many of which are associated with serious, sometimes fatal disease(s). When a tick travel souvenir is presented in the clinic, the risk must be immediately assessed by identifying the tick in question, ascertaining its disease vector status and determining if there has been the opportunity for the transfer of potential pathogens. This short review on identification of disease vector ticks and aspects of blood feeding and disease transmission includes the results of an examination of 59 specimens removed from UK domestic travellers and international travellers between 2002 and 2010. Sixteen tick species belonging to six genera were recorded and almost all showed evidence of blood feeding, which appears to contradict the view that because of their size, adult ticks are found early and therefore present an insignificant risk. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Generalization of concurrence vectors

    International Nuclear Information System (INIS)

    Yu Changshui; Song Heshan

    2004-01-01

    In this Letter, based on the generalization of concurrence vectors for bipartite pure state with respect to employing tensor product of generators of the corresponding rotation groups, we generalize concurrence vectors to the case of mixed states; a new criterion of separability of multipartite pure states is given out, for which we define a concurrence vector; we generalize the vector to the case of multipartite mixed state and give out a good measure of free entanglement

  20. Vector Network Coding

    OpenAIRE

    Ebrahimi, Javad; Fragouli, Christina

    2010-01-01

    We develop new algebraic algorithms for scalar and vector network coding. In vector network coding, the source multicasts information by transmitting vectors of length L, while intermediate nodes process and combine their incoming packets by multiplying them with L X L coding matrices that play a similar role as coding coefficients in scalar coding. Our algorithms for scalar network jointly optimize the employed field size while selecting the coding coefficients. Similarly, for vector co...

  1. Vector Network Coding Algorithms

    OpenAIRE

    Ebrahimi, Javad; Fragouli, Christina

    2010-01-01

    We develop new algebraic algorithms for scalar and vector network coding. In vector network coding, the source multicasts information by transmitting vectors of length L, while intermediate nodes process and combine their incoming packets by multiplying them with L x L coding matrices that play a similar role as coding c in scalar coding. Our algorithms for scalar network jointly optimize the employed field size while selecting the coding coefficients. Similarly, for vector coding, our algori...

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  4. 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.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. 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

  8. Convexity and Marginal Vectors

    NARCIS (Netherlands)

    van Velzen, S.; Hamers, H.J.M.; Norde, H.W.

    2002-01-01

    In this paper we construct sets of marginal vectors of a TU game with the property that if the marginal vectors from these sets are core elements, then the game is convex.This approach leads to new upperbounds on the number of marginal vectors needed to characterize convexity.An other result is that

  9. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan

    2015-01-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  10. 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...

  11. Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model

    International Nuclear Information System (INIS)

    Freytag, Svend O.; Paielli, Dell; Wing, Mark; Rogulski, Ken; Brown, Steve; Kolozsvary, Andy; Seely, John; Barton, Ken; Dragovic, Alek; Kim, Jae Ho

    2002-01-01

    , trimodal therapy was associated with little toxicity. A comparison of the major histopathologic findings among the treatment groups indicated that most of the locoregional (prostate, seminal vesicles, urinary bladder) pathology was attributable to the combined effects of the Ad5-CD/TKrep vector and EBRT and that the prodrugs contributed little to this effect. Importantly, trimodal therapy did not exacerbate inflammation of the rectum and intestines beyond that of EBRT alone. Conclusion: Together, the results support the thesis that replication-competent adenovirus-mediated double suicide gene therapy may be a safe and effective adjuvant to EBRT and provide a sound scientific rationale for human trials

  12. 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.

    Science.gov (United States)

    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.

  13. Rotations with Rodrigues' vector

    International Nuclear Information System (INIS)

    Pina, E

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears to be a fundamental matrix that is used to express the components of the angular velocity, the rotation matrix and the angular momentum vector. The Hamiltonian formalism of rotational dynamics in terms of this vector uses the same matrix. The quantization of the rotational dynamics is performed with simple rules if one uses Rodrigues' vector and similar formal expressions for the quantum operators that mimic the Hamiltonian classical dynamics.

  14. Derivation of a triple mosaic adenovirus for cancer gene therapy.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

    Science.gov (United States)

    Kanao, Megumi; Kanda, Hirotsugu; Huang, Wan; Liu, Shue; Yi, Hyun; Candiotti, Keith A; Lubarsky, David A; Levitt, Roy C; Hao, Shuanglin

    2015-06-01

    Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPβ was tested using immunohistochemistry. In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the

  17. 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.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Supergravity inspired vector curvaton

    International Nuclear Information System (INIS)

    Dimopoulos, Konstantinos

    2007-01-01

    It is investigated whether a massive Abelian vector field, whose gauge kinetic function is growing during inflation, can be responsible for the generation of the curvature perturbation in the Universe. Particle production is studied and it is shown that the vector field can obtain a scale-invariant superhorizon spectrum of perturbations with a reasonable choice of kinetic function. After inflation the vector field begins coherent oscillations, during which it corresponds to pressureless isotropic matter. When the vector field dominates the Universe, its perturbations give rise to the observed curvature perturbation following the curvaton scenario. It is found that this is possible if, after the end of inflation, the mass of the vector field increases at a phase transition at temperature of order 1 TeV or lower. Inhomogeneous reheating, whereby the vector field modulates the decay rate of the inflaton, is also studied

  2. 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...

  3. 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

  4. Custodial vector model

    Science.gov (United States)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan; Frandsen, Mads T.; Hapola, Tuomas; Sannino, Francesco

    2015-07-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a S U (2 )L×S U (2 )R spectral global symmetry. This symmetry partially protects the electroweak S parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum and interactions with the standard model fields lead to distinct signatures at the LHC in the diboson, dilepton, and associated Higgs channels.

  5. Vector Differential Calculus

    OpenAIRE

    HITZER, Eckhard MS

    2002-01-01

    This paper treats the fundamentals of the vector differential calculus part of universal geometric calculus. Geometric calculus simplifies and unifies the structure and notation of mathematics for all of science and engineering, and for technological applications. In order to make the treatment self-contained, I first compile all important geometric algebra relationships,which are necesssary for vector differential calculus. Then differentiation by vectors is introduced and a host of major ve...

  6. Implicit Real Vector Automata

    Directory of Open Access Journals (Sweden)

    Jean-François Degbomont

    2010-10-01

    Full Text Available This paper addresses the symbolic representation of non-convex real polyhedra, i.e., sets of real vectors satisfying arbitrary Boolean combinations of linear constraints. We develop an original data structure for representing such sets, based on an implicit and concise encoding of a known structure, the Real Vector Automaton. The resulting formalism provides a canonical representation of polyhedra, is closed under Boolean operators, and admits an efficient decision procedure for testing the membership of a vector.

  7. Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells

    International Nuclear Information System (INIS)

    Zheng Changyu; O'Connell, Brian C.; Baum, Bruce J.

    2003-01-01

    Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event

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

    Science.gov (United States)

    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.

  9. Vectorized Monte Carlo

    International Nuclear Information System (INIS)

    Brown, F.B.

    1981-01-01

    Examination of the global algorithms and local kernels of conventional general-purpose Monte Carlo codes shows that multigroup Monte Carlo methods have sufficient structure to permit efficient vectorization. A structured multigroup Monte Carlo algorithm for vector computers is developed in which many particle events are treated at once on a cell-by-cell basis. Vectorization of kernels for tracking and variance reduction is described, and a new method for discrete sampling is developed to facilitate the vectorization of collision analysis. To demonstrate the potential of the new method, a vectorized Monte Carlo code for multigroup radiation transport analysis was developed. This code incorporates many features of conventional general-purpose production codes, including general geometry, splitting and Russian roulette, survival biasing, variance estimation via batching, a number of cutoffs, and generalized tallies of collision, tracklength, and surface crossing estimators with response functions. Predictions of vectorized performance characteristics for the CYBER-205 were made using emulated coding and a dynamic model of vector instruction timing. Computation rates were examined for a variety of test problems to determine sensitivities to batch size and vector lengths. Significant speedups are predicted for even a few hundred particles per batch, and asymptotic speedups by about 40 over equivalent Amdahl 470V/8 scalar codes arepredicted for a few thousand particles per batch. The principal conclusion is that vectorization of a general-purpose multigroup Monte Carlo code is well worth the significant effort required for stylized coding and major algorithmic changes

  10. Vectors and their applications

    CERN Document Server

    Pettofrezzo, Anthony J

    2005-01-01

    Geared toward undergraduate students, this text illustrates the use of vectors as a mathematical tool in plane synthetic geometry, plane and spherical trigonometry, and analytic geometry of two- and three-dimensional space. Its rigorous development includes a complete treatment of the algebra of vectors in the first two chapters.Among the text's outstanding features are numbered definitions and theorems in the development of vector algebra, which appear in italics for easy reference. Most of the theorems include proofs, and coordinate position vectors receive an in-depth treatment. Key concept

  11. Symbolic computer vector analysis

    Science.gov (United States)

    Stoutemyer, D. R.

    1977-01-01

    A MACSYMA program is described which performs symbolic vector algebra and vector calculus. The program can combine and simplify symbolic expressions including dot products and cross products, together with the gradient, divergence, curl, and Laplacian operators. The distribution of these operators over sums or products is under user control, as are various other expansions, including expansion into components in any specific orthogonal coordinate system. There is also a capability for deriving the scalar or vector potential of a vector field. Examples include derivation of the partial differential equations describing fluid flow and magnetohydrodynamics, for 12 different classic orthogonal curvilinear coordinate systems.

  12. On the mechanism of arginine requirement for adenovirus synthesis

    International Nuclear Information System (INIS)

    Plaat, D.; Weber, J.

    1979-01-01

    The effects of arginine deprivation on the synthesis and processing of viral proteins and the assembly of incomplete and complete virions were studied during infection with human adenovirus type 2. Arginine deprivation greatly reduced the synthesis of all viral proteins, particularly the precursor to core protein VII. The inhibition was completely reversible by the addition of arginine to the medium. Arginine deprivation between 7 and 20 hours post-infection inhibited the processing of PVII to VII, suggesting that PVII is not cleaved autocatalytically. The assembly of incomplete virions was sensitive to arginine deprivation only prior to 20 hours, while the assembly of complete virions was dependent on the continuous presence of arginine. This observation supports the hypothesis that incomplete virions are precursors of complete virions. The experiments on the PVII-specific endoprotease activity showed that arginine deprivation caused only slight reduction in the in vitro activity, although no activity was observed in vivo. The present results lead to the hypothesis that arginine deficiency inhibits the synthesis of a functional protein essential for virion maturation, other than the synthesis of processing of PVII. (author)

  13. Determination of the transforming activities of adenovirus oncogenes.

    Science.gov (United States)

    Speiseder, Thomas; Nevels, Michael; Dobner, Thomas

    2014-01-01

    The last 50 years of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells, human amniotic fluid cells and athymic nude mice.

  14. Construction of an infectious clone of human adenovirus type 41.

    Science.gov (United States)

    Chen, Duo-Ling; Dong, Liu-Xin; Li, Meng; Guo, Xiao-Juan; Wang, Min; Liu, Xin-Feng; Lu, Zhuo-Zhuang; Hung, Tao

    2012-07-01

    Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

  15. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    2015-11-01

    Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Suppression of Oncolytic Adenovirus-Mediated Hepatotoxicity by Liver-Specific Inhibition of NF-κB

    Directory of Open Access Journals (Sweden)

    Mitsuhiro Machitani

    2017-12-01

    Full Text Available Telomerase-specific replication-competent adenoviruses (Ads, i.e., TRADs, which possess an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, are promising agents for cancer treatment. However, even though oncolytic Ads, including TRAD, are intratumorally administered, they are disseminated from the tumor to systemic circulation, causing concern about oncolytic Ad-mediated hepatotoxicity (due mainly to leaky expression of Ad genes in liver. We reported that inhibition of nuclear factor-κB (NF-κB leads to the suppression of replication-incompetent Ad vector-mediated hepatotoxicity via reduction of the leaky expression of Ad genes in liver. Here, to develop a TRAD with an improved safety profile, we designed a TRAD that carries a liver-specific promoter-driven dominant-negative IκBα (DNIκBα expression cassette (TRAD-DNIκBα. Compared with a conventional TRAD, TRAD-DNIκBα showed hepatocyte-specific inhibition of NF-κB signaling and significantly reduced Ad gene expression and replication in the normal human hepatocyte cell line. TRAD-induced hepatotoxicity was largely suppressed in mice following intravenous administration of TRAD-DNIκBα. However, the replication profiles and oncolytic activities of TRAD-DNIκBα were comparable with those of the conventional TRAD in human non-hepatic tumor cells. These results indicate that oncolytic Ads containing the liver-specific DNIκBα expression cassette have improved safety profiles without inhibiting oncolytic activities.

  19. 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

  20. 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.

  1. Taxonomy proposal for Old World monkey adenoviruses: characterisation of several non-human, non-ape primate adenovirus lineages.

    Science.gov (United States)

    Pantó, Laura; Podgorski, Iva I; Jánoska, Máté; Márkó, Orsolya; Harrach, Balázs

    2015-12-01

    A species classification regarding Old World monkey adenoviruses is proposed. We determined the nucleotide sequences of PCR-amplified fragments from the genes of the IVa2, DNA-dependent DNA polymerase, penton base, and hexon proteins from every simian adenovirus (SAdV) serotype that originated from Old World monkeys for which the full genome sequence had not yet been published. We confirmed that the majority of Old Word monkey SAdVs belong to two previously established species. Interestingly, one is the most recently established human AdV species, Human mastadenovirus G, which includes a single human virus, HAdV-52, as well as SAdV-1, -2, -7, -11, -12, and -15. The other approved species, Simian mastadenovirus A includes SAdV-3, -4, -6, -9, -10, -14, and -48. Several SAdVs (SAdV-5, -8, -49, -50) together with baboon AdV-1 and rhesus monkey AdV strains A1139, A1163, A1173, A1258, A1285, A1296, A1312, A1327 and A1335 have been proposed to be classified into an additional species, Simian mastadenovirus B. Another proposed species, Simian mastadenovirus C has been described for SAdV-19, baboon AdV-2/4 and -3. Our study revealed the existence of four additional AdV lineages. The corresponding new candidate species are Simian mastadenovirus D (for SAdV-13), Simian mastadenovirus E (for SAdV-16), Simian mastadenovirus F (for SAdV-17 and -18), and Simian mastadenovirus G (for SAdV-20). Several biological and genomic properties, such as the host origin, haemagglutination profile, number of fibre genes, and G+C content of the genome, strongly support this classification. Three SAdV strains originating from the American Type Culture Collection turned out to be mixtures of at least two virus types, either of the same species (SAdV-12 and -15 types from Human mastadenovirus G) or of two different species (SAdV-5 types from Simian mastadenovirus B and Human mastadenovirus G).

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  5. 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.

  6. 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

  7. 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

  8. Radioecology. Transfers of radioelements

    International Nuclear Information System (INIS)

    Foulquier, L.

    2002-01-01

    The study of the cycle of radioelements in the environment requires the measurement of the radionuclides present in all parts of the ecosystems. The knowledge of the mechanisms of radioactive pollutant transfers and of the kinetics of the exchanges between a source term, the vectors and the constituents of the biosphere represents heart of the work of radio-ecologists. This article describes briefly the techniques used for the measurement of radionuclides in the environment and for the study of their physical dispersion mechanisms. Then, it treats more carefully of the transfer mechanisms in different environments: 1 - tools for the evaluation of transfers: metrology, atmospheric and liquid dispersion phenomena; 2 - processes of radioelement transfers: transfers in aquatic ecosystems, transfers in terrestrial environment. (J.S.)

  9. Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

    Science.gov (United States)

    Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J; Farness, Peggy; Avanzini, Jenny B; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J; Mayall, Tim

    2012-01-01

    Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

  10. Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes.

    Science.gov (United States)

    Holman, David H; Wang, Danher; Raviprakash, Kanakatte; Raja, Nicholas U; Luo, Min; Zhang, Jianghui; Porter, Kevin R; Dong, John Y

    2007-02-01

    Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

  11. Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Jeff Alexander

    Full Text Available Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4 vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn. Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  14. Vector-Vector Scattering on the Lattice

    Science.gov (United States)

    Romero-López, Fernando; Urbach, Carsten; Rusetsky, Akaki

    2018-03-01

    In this work we present an extension of the LüScher formalism to include the interaction of particles with spin, focusing on the scattering of two vector particles. The derived formalism will be applied to Scalar QED in the Higgs Phase, where the U(1) gauge boson acquires mass.

  15. 5-Fluorouracil-related enhancement of adenoviral infection is Coxsackievirus-adenovirus receptor independent and associated with morphological changes in lipid membranes

    Science.gov (United States)

    Cabrele, Chiara; Vogel, Mandy; Piso, Pompiliu; Rentsch, Markus; Schröder, Josef; Jauch, Karl W; Schlitt, Hans J; Beham, Alexander

    2006-01-01

    AIM: To evaluate the mechanism underlying the effects of 5-Fluorouracil (5-FU) on adenoviral infection. METHODS: Low and high Coxsackievirus-Adenovirus Receptor (CAR) expressing human colon carcinoma cell lines were treated with 5-FU and two E1-deleted adenoviral constructs, one transferring GFP (Ad/CMV-GFP) the other bax (Ad/CEA-bax). The number of infected cells were monitored by GFP expression. To evaluate the effects of 5-FU in a receptor free system, Ad/GFP were encapsulated in liposomes and treated with 5-FU. Ad/GFP release was estimated with PCR and infection of 293 cells with the supernatant. Electron microscopy of the Ad5-GFP-liposome complex was made to investigate morphological changes of the liposomes after 5-FU. RESULTS: Infection rates of all cell lines increased from 50% to 98% with emerging 5-FU concentrations. The enhanced viral uptake was independent of the CAR expression. Additionally, 5-FU treated liposomes released 2-2.5 times more adenoviruses. Furthermore, 5-FU-treated liposomes appeared irregular and porous-like. CONCLUSION: adenoviral uptake is enhanced in the presence of 5-FU irrespective of CAR and is associated with morphological changes in membranes making the combination of both a promising option in gene therapy. PMID:16937527

  16. Selection vector filter framework

    Science.gov (United States)

    Lukac, Rastislav; Plataniotis, Konstantinos N.; Smolka, Bogdan; Venetsanopoulos, Anastasios N.

    2003-10-01

    We provide a unified framework of nonlinear vector techniques outputting the lowest ranked vector. The proposed framework constitutes a generalized filter class for multichannel signal processing. A new class of nonlinear selection filters are based on the robust order-statistic theory and the minimization of the weighted distance function to other input samples. The proposed method can be designed to perform a variety of filtering operations including previously developed filtering techniques such as vector median, basic vector directional filter, directional distance filter, weighted vector median filters and weighted directional filters. A wide range of filtering operations is guaranteed by the filter structure with two independent weight vectors for angular and distance domains of the vector