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Sample records for adenovirus ad vector

  1. Influence of coagulation factor x on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors.

    PubMed

    Greig, Jenny A; Buckley, Suzanne Mk; Waddington, Simon N; Parker, Alan L; Bhella, David; Pink, Rebecca; Rahim, Ahad A; Morita, Takashi; Nicklin, Stuart A; McVey, John H; Baker, Andrew H

    2009-10-01

    The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon-containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5.

  2. Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

    PubMed

    Habib, Nagy A; Mitry, Ragai; Seth, Prem; Kuppuswamy, Mohan; Doronin, Konstantin; Toth, Karoly; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

    2002-08-01

    The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

  3. Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5.

    PubMed

    Holterman, Lennart; Vogels, Ronald; van der Vlugt, Remko; Sieuwerts, Martijn; Grimbergen, Jos; Kaspers, Jorn; Geelen, Eric; van der Helm, Esmeralda; Lemckert, Angelique; Gillissen, Gert; Verhaagh, Sandra; Custers, Jerome; Zuijdgeest, David; Berkhout, Ben; Bakker, Margreet; Quax, Paul; Goudsmit, Jaap; Havenga, Menzo

    2004-12-01

    A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.

  4. Adenovirus serotype 35 vector-induced innate immune responses in dendritic cells derived from wild-type and human CD46-transgenic mice: Comparison with a fiber-substituted Ad vector containing fiber proteins of Ad serotype 35.

    PubMed

    Sakurai, Fuminori; Nakashima, Kazuko; Yamaguchi, Tomoko; Ichinose, Takako; Kawabata, Kenji; Hayakawa, Takao; Mizuguchi, Hiroyuki

    2010-12-01

    Recently, much attention has focused on replication-incompetent adenovirus (Ad) vectors containing fiber proteins derived from species B Ad serotype 35 (Ad35) (Ad5F35) and Ad vectors fully constructed from Ad35 as vaccine vectors expressing antigens. However, differences in the transduction properties, including the induction of innate immunity, of Ad5F35 and Ad35 vectors have not been properly and fully examined, partly because the transduction properties of these Ad vectors should be evaluated using nonhuman primates or human CD46-transgenic (CD46TG) mice, which ubiquitously express the primary receptor of Ad35, human CD46, in a pattern similar to that of humans. In the present study, we evaluated innate immune responses of mouse dendritic cells (mDCs) derived from bone marrow cells of wild-type (WT) and CD46TG mice following transduction with Ad serotype 5 (Ad5), fiber-substituted Ad5F35, or Ad35 vectors. Ad5F35 and Ad35 vectors mediated more efficient transduction in mDCs derived from CD46TG mice (CD46TG-mDCs) than did Ad5 vectors. Upregulation of costimulatory molecules and inflammatory cytokine induction by Ad5F35 and Ad35 vectors were significantly higher than those by Ad5 vectors in CD46TG-mDCs. However, the induction properties of the innate immune responses were different between Ad5F35 and Ad35 vectors. Ad35 vectors induced higher levels of costimulatory molecule expression and inflammatory cytokine production than did Ad5F35 vectors in CD46TG-mDCs. Furthermore, intravenous administration of Ad35 vectors in WT and CD46TG mice resulted in higher levels of serum interleukin (IL)-6 and IL-12 compared with administration of Ad5F35 vectors, which exhibited almost mock-transduced levels of these inflammatory cytokines. This study indicates that innate immune responses by Ad35 and Ad5F35 vectors are distinct even although both Ad vectors recognize human CD46 as a receptor.

  5. Differential immunogenicity between HAdV-5 and chimpanzee adenovirus vector ChAdOx1 is independent of fiber and penton RGD loop sequences in mice

    PubMed Central

    Dicks, Matthew D. J.; Spencer, Alexandra J.; Coughlan, Lynda; Bauza, Karolis; Gilbert, Sarah C.; Hill, Adrian V. S.; Cottingham, Matthew G.

    2015-01-01

    Replication defective adenoviruses are promising vectors for the delivery of vaccine antigens. However, the potential of a vector to elicit transgene-specific adaptive immune responses is largely dependent on the viral serotype used. HAdV-5 (Human adenovirus C) vectors are more immunogenic than chimpanzee adenovirus vectors from species Human adenovirus E (ChAdOx1 and AdC68) in mice, though the mechanisms responsible for these differences in immunogenicity remain poorly understood. In this study, superior immunogenicity was associated with markedly higher levels of transgene expression in vivo, particularly within draining lymph nodes. To investigate the viral factors contributing to these phenotypes, we generated recombinant ChAdOx1 vectors by exchanging components of the viral capsid reported to be principally involved in cell entry with the corresponding sequences from HAdV-5. Remarkably, pseudotyping with the HAdV-5 fiber and/or penton RGD loop had little to no effect on in vivo transgene expression or transgene-specific adaptive immune responses despite considerable species-specific sequence heterogeneity in these components. Our results suggest that mechanisms governing vector transduction after intramuscular administration in mice may be different from those described in vitro. PMID:26576856

  6. Vaccination to conserved influenza antigens in mice using a novel Simian adenovirus vector, PanAd3, derived from the bonobo Pan paniscus.

    PubMed

    Vitelli, Alessandra; Quirion, Mary R; Lo, Chia-Yun; Misplon, Julia A; Grabowska, Agnieszka K; Pierantoni, Angiolo; Ammendola, Virginia; Price, Graeme E; Soboleski, Mark R; Cortese, Riccardo; Colloca, Stefano; Nicosia, Alfredo; Epstein, Suzanne L

    2013-01-01

    Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.

  7. Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector, PanAd3, Derived from the Bonobo Pan paniscus

    PubMed Central

    Vitelli, Alessandra; Quirion, Mary R.; Lo, Chia-Yun; Misplon, Julia A.; Grabowska, Agnieszka K.; Pierantoni, Angiolo; Ammendola, Virginia; Price, Graeme E.; Soboleski, Mark R.; Cortese, Riccardo; Colloca, Stefano; Nicosia, Alfredo; Epstein, Suzanne L.

    2013-01-01

    Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines. PMID:23536756

  8. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

    Zhou, Xiangyang; Xiang, Zhiquan; Ertl, Hildegund C J

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies.

  9. [Construction of replication-deficient recombinant adenovirus vector with hTFPI-2 gene by AdMax system and expression in U937 monocytes in vitro].

    PubMed

    Pan, Junjie; Shi, Haiming; Luo, Xinping; Ma, Duan; Liang, Wang; Zhang, Jin; Zhu, Jun; Li, Jian

    2011-04-01

    We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2 (hTFPI-2) gene by AdMax system in HEK293 cells. Firstly, we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI. After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector, we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2. It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing. Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxdeltaE1,3Cre by liposome lipofectamine2000, and finished the package of recombinant adenovirus Ad-hTFPI-2. The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2. Furthermore, we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification. The titre was 0.931 x 10(12) pfu/ml. Finally, we infected U937 monocytes by purified Ad-hTFPI-2, and determined the infection efficiency and the TFPI-2's level and activity. The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%. After infected by Ad-hTFPI-2, the TFPI-2's level in supernatant increased about 7 fold. Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin. The recombinant adenovirus with the hTFPI-2 gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in antiatherosclerosis.

  10. An Oncotropic Adenovirus Vector System for Breast Cancer Treatment

    DTIC Science & Technology

    2005-09-01

    AD Award Number: DAMD17-03-1-0629 TITLE: An Oncotropic Adenovirus Vector System for Breast Cancer Treatment PRINCIPAL INVESTIGATOR: Igor P. Dmitriev...Aug 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An Oncotropic Adenovirus Vector System for Breast Cancer Treatment 5b. GRANT NUMBER DAMD17-03-1...epithelial cells, the origin of most human cancers. However, realization of the full potential of Ad vectors for targeted cancer treatment is currently

  11. Viral Vector Production: Adenovirus.

    PubMed

    Kim, Julius W; Morshed, Ramin A; Kane, J Robert; Auffinger, Brenda; Qiao, Jian; Lesniak, Maciej S

    2016-01-01

    Adenoviral vectors have proven to be valuable resources in the development of novel therapies aimed at targeting pathological conditions of the central nervous system, including Alzheimer's disease and neoplastic brain lesions. Not only can some genetically engineered adenoviral vectors achieve remarkably efficient and specific gene delivery to target cells, but they also may act as anticancer agents by selectively replicating within cancer cells.Due to the great interest in using adenoviral vectors for various purposes, the need for a comprehensive protocol for viral vector production is especially apparent. Here, we describe the process of generating an adenoviral vector in its entirety, including the more complex process of adenoviral fiber modification to restrict viral tropism in order to achieve more efficient and specific gene delivery.

  12. 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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  13. Chimpanzee Adenovirus Vector Ebola Vaccine.

    PubMed

    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

  14. [Gene engineering of the adenovirus vector].

    PubMed

    Kondo, Saki; Terashima, Miho; Fukuda, Hiromitsu; Saito, Izumu; Kanegae, Yumi

    2007-06-01

    The adenovirus vector is very attractive tool not only for the gene therapy but also for the basic sciences. However, because a construction method of this vector had been complex, only limited scientists had constructed and enjoyed the benefits. Recently, various methods were developed and the researchers came to be able to choose an efficient method, which is the COS-TPC method, or a concise procedure, which is the intact-genome transfection method (in vitro ligation method). Here we described not only these methods but also new method to construct the various Ads simultaneously using the recombinase-mediated cassette exchange (RMCE) by the site-specific recombinase. And also we want to refer the possibility to the worth of the vector, especially the vector of the expression-switch.

  15. Transductional targeting of adenovirus vectors for gene therapy

    PubMed Central

    Glasgow, JN; Everts, M; Curiel, DT

    2007-01-01

    Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions. PMID:16439993

  16. Immunogenicity of the recombinant adenovirus type-5 vector -based Ebola vaccine (Ad5-EBOV) expressing the glycoprotein of the 2014 epidemic strain in mice.

    PubMed

    Wang, Ling; Liu, Jing Jing; Kong, Yan; Hou, Li Hua; Li, Yu Hua

    2017-08-10

    The 2014 Ebola outbreak in West Africa has brought great threat to the public health worldwide. Development of Ebola vaccine is urgent. A novel recombinant adenovirus type-5 vector-based Ebola vaccine (Ad5-EBOV) based on the 2014 Zaire Guinea epidemic strain was developed in China. A good safety profile and robust immune response elicited by Ad5-EBOV were confirmed in Phase I and Phase II clinical trial. However, clinical studies of Ebola vaccine are still at an early stage and no solid efficacy data for human beings yet. For efficacy evaluation and quality control of Ad5-EBOV, the cellular and humoral immune responses were examined at different time points in BALB/c mice which were vaccinated with the Ad5-EBOV. EBOV GP-specific T-cell responses were detected early in the first week, increased overtime, reached the peak at week 4 and lasted to week 6 by ELISpot and Flow cytometric analysis. High titer of EBOV glycoprotein-specific antibody was found at week 1 at 1783(geometric mean, GM), peaked at week 10 at 26,214(GM), and lasted to six months at 1351(GM). The titer of neutralizing antibody based on pseudovirus also increased overtime, peaked at 1:16 in one mouse and 1:8 in nine mice at week 6, then decreased to zero at week 12. These results suggest that BALB/c mice can be used to evaluate the effectiveness of Ad5-EBOV, while cellular immune response and humoral immune response can be used as indicators for the evaluation of vaccine effectiveness. Determination of methods and indicators as soon as possible is critical and valuable for efficacy evaluation of Ebola vaccine, and is expected to provide an important guarantee for quality control of Ad5-EBOV.

  17. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    SciTech Connect

    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.

    2014-11-19

    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. Furthermore, 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. 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.

  18. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    DOE PAGES

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; ...

    2014-11-19

    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. Furthermore, 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. 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 tomore » 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.« less

  19. An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism.

    PubMed

    Glasgow, Joel N; Kremer, Eric J; Hemminki, Akseli; Siegal, Gene P; Douglas, Joanne T; Curiel, David T

    2004-06-20

    Many clinically relevant tissues are refractory to Ad5 transduction because of negligible levels of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of Ad vectors that display CAR-independent tropism could lead directly to therapeutic gain. The Toronto strain of canine adenovirus type 2 (CAV2) exhibits native tropism that is augmented by, but not fully dependent upon, CAR for cellular transduction. We hypothesized that an Ad5 vector containing the nonhuman CAV2 knob would provide expanded tropism and constructed Ad5Luc1-CK, an E1-deleted Ad5 vector encoding the fiber knob domain from CAV2. Ad5Luc1-CK gene delivery to CAR-deficient cells was augmented up to 30-fold versus the Ad5 control vector, and correlated with increased cell surface binding. Further, we confirmed the importance of cellular integrins to Ad5Luc1-CK transduction. Herein, we present the rationale, design, purification, and characterization of a novel tropism modified, infectivity-enhanced Ad vector.

  20. Adenovirus Vectors Targeting Distinct Cell Types in the Retina

    PubMed Central

    Sweigard, J. Harry; Cashman, Siobhan M.

    2010-01-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5ΔRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5ΔRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5ΔRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5ΔRGD vectors. PMID:19892875

  1. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine

    USDA-ARS?s Scientific Manuscript database

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding a H7 hemagglutinin gene from a low pathogenic North American isolate (AdChNY94.H7). Chickens vaccinate...

  2. Characterization of human adenovirus 35 and derivation of complex vectors

    PubMed Central

    2010-01-01

    Background Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery. Results Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors. Conclusions The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes. PMID:20959004

  3. A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

    PubMed Central

    Farrow, Anitra L.; Peng, Binghao J.; Gu, Linlin; Krendelchtchikov, Alexandre; Matthews, Qiana L.

    2016-01-01

    Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease. PMID:26978385

  4. Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

    PubMed

    Gao, Dong-sheng; Li, Xiao-jing; Wan, Wen-yan; Li, Hong-jie; Wang, Xiao-xue; Yang, Xia; Li, Yong-tao; Chang, Hong-tao; Chen, Lu; Wang, Chuan-qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

  5. Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors.

    PubMed

    Seregin, Sergey S; Amalfitano, Andrea

    2009-12-01

    Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.

  6. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

    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.

  7. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

    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. PMID:25876176

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

    SciTech Connect

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    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.

  9. Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses

    PubMed Central

    Crosby, Catherine M.; Matchett, William E.; Anguiano-Zarate, Stephanie S.; Parks, Christopher A.; Weaver, Eric A.; Pease, Larry R.; Webby, Richard J.

    2016-01-01

    ABSTRACT Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines. IMPORTANCE Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold

  10. Production of high-capacity adenovirus vectors.

    PubMed

    Kreppel, Florian

    2014-01-01

    High-capacity adenoviral vectors (HC-Ad), also known as "helper-dependent" (HD-Ad), "gutless", "gutted", or "third-generation" Ad vectors, are devoid of all viral coding sequences and have shown promising potential for a wide variety of different applications-from classic gene therapy to genetic vaccination and tumor treatment. However, compared to first-generation adenoviral vectors their production is more complex and requires specific in-depth knowledge. This chapter delivers a detailed protocol for the successful production of HC-Ad vectors to high titers.

  11. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

    PubMed Central

    Stevenson, S C; Rollence, M; Marshall-Neff, J; McClelland, A

    1997-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange

  12. Recent Advances in Preclinical Developments Using Adenovirus Hybrid Vectors.

    PubMed

    Ehrke-Schulz, Eric; Zhang, Wenli; Gao, Jian; Ehrhardt, Anja

    2017-10-01

    Adenovirus (Ad)-based vectors are efficient gene-transfer vehicles to deliver foreign DNA into living organisms, offering large cargo capacity and low immunogenicity and genotoxicity. As Ad shows low integration rates of their genomes into host chromosomes, vector-derived gene expression decreases due to continuous cell cycling in regenerating tissues and dividing cell populations. To overcome this hurdle, adenoviral delivery can be combined with mechanisms leading to maintenance of therapeutic DNA and long-term effects of the desired treatment. Several hybrid Ad vectors (AdV) exploiting various strategies for long-term treatment have been developed and characterized. This review summarizes recent developments of preclinical approaches using hybrid AdVs utilizing either the Sleeping Beauty transposase system for somatic integration into host chromosomes or designer nucleases, including transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease for permanent gene editing. Further options on how to optimize these vectors further are discussed, which may lead to future clinical applications of these versatile gene-therapy tools.

  13. Adenovirus dodecahedron, a new vector for human gene transfer.

    PubMed

    Fender, P; Ruigrok, R W; Gout, E; Buffet, S; Chroboczek, J

    1997-01-01

    Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

  14. Modification of pIX or hexon based on fiberless Ad vectors is not effective for targeted Ad vectors.

    PubMed

    Kurachi, Shinnosuke; Koizumi, Naoya; Tashiro, Katsuhisa; Sakurai, Haruna; Sakurai, Fuminori; Kawabata, Kenji; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki

    2008-04-07

    Adenovirus (Ad) vector application in gene therapy is limited by its naïve tropism. We previously developed protein IX (pIX)-modified and hexon-modified Ad vectors in order to alter Ad vector tropism. However, these modified Ad vectors failed to infect cells with the foreign ligands displayed in the pIX or hexon. We hypothesized that steric hindrance by fiber proteins might have prevented the ligand-mediated transduction, as fibers are the outmost capsid proteins of Ad vectors. Therefore, we generated a series of fiberless Ad vectors and investigated their gene expression properties. Unexpectively, however, pIX- or hexon-modified fiberless Ad vector did not achieve any gene expression (the gene expression level by these vectors was similar to the background level). These results might be caused by the fact that the fiberless particles were weaker against physical burdens. To the best of our knowledge, this study is the first reported attempt to develop fiberless Ad vectors containing foreign ligands in the pIX or hexon region. The drawback of the lower stability of fiberless Ad vectors must be overcome to develop targeted Ad vectors based on such vectors. This study could provide basic information for the development of effective targeted Ad vectors.

  15. Adenovirus vector-based vaccines for human immunodeficiency virus type 1.

    PubMed

    Barouch, Dan H; Nabel, Gary J

    2005-02-01

    Recombinant adenovirus (rAd) vectors have received considerable attention for gene therapy because of their high transduction efficiency. However, recombinant gene expression from rAd vectors elicits rapid and potent immune responses to foreign transgene products. Such immunogenicity limits the duration of transgene expression and poses a major challenge to the use of rAd vectors for gene therapy. In contrast, the inherent immunogenicity of these vectors is a desirable feature for vaccine development. The immunogenicity and protective efficacy of rAd vector-based vaccines have now been demonstrated in a number of animal models, and rAd vaccines for a variety of pathogens are currently being explored in early-phase clinical trials. In this review, we describe progress in the development of rAd vector-based vaccines with a focus on human immunodeficiency virus type 1.

  16. Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

    PubMed

    Belousova, Natalya; Mikheeva, Galina; Xiong, Chiyi; Stagg, Loren J; Gagea, Mihai; Fox, Patricia S; Bassett, Roland L; Ladbury, John E; Braun, Michael B; Stehle, Thilo; Li, Chun; Krasnykh, Victor

    2016-08-16

    Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.

  17. Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    PubMed Central

    Belousova, Natalya; Mikheeva, Galina; Xiong, Chiyi; Stagg, Loren J.; Gagea, Mihai; Fox, Patricia S.; Bassett, Roland L.; Ladbury, John E.; Braun, Michael B.; Stehle, Thilo; Li, Chun; Krasnykh, Victor

    2016-01-01

    Unique molecular properties of species D adenoviruses (Ads)—the most diverse yet underexplored group of Ads—have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies. PMID:27462785

  18. Characterization of Species C Human Adenovirus Serotype 6 (Ad6)

    PubMed Central

    Weaver, Eric A.; Khare, Reeti; Hillestad, Mathew L.; Palmer, Donna; Ng, Philip; Barry, Michael A.

    2010-01-01

    Adenovirus serotype (Ad5) is the most studied Ad. Ad1, 2, and 6 are also members of species C Ad and are presumed to have biologies similar to Ad5. In this work, we have compared the ability of Ad1, 2, 5, and 6 to infect liver and muscle after intravenous and intramuscular injection. We found that Ad6 was surprisingly the most potent at liver gene delivery and that Ad1 and Ad2 were markedly weaker than Ad5 and 6. To understand these differences, we sequenced the Ad6 genome. This revealed that the Ad6 fiber protein is surprisingly three shaft repeats shorter than the others which may explain differences in virus infectivity in vitro, but not in the liver. Comparison of hexon hypervariable regions (HVRs) suggests that the higher transduction by Ad5 and 6 as compared to Ad1 and 2 may be related to differences in charge and length. PMID:21251688

  19. Packaging of an AAV vector encoding human acid alpha-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector.

    PubMed

    Sun, Baodong; Chen, Y-T; Bird, Andrew; Xu, Fang; Hou, Yang-Xun; Amalfitano, Andrea; Koeberl, Dwight D

    2003-04-01

    We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid alpha-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10(10) AAV vector particles. These modifications resulted in approximately 30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid alpha-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.

  20. Construction, production, and purification of recombinant adenovirus vectors.

    PubMed

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel

    2014-01-01

    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  1. Adenovirus vector delivery stimulates natural killer cell recognition

    PubMed Central

    Tomasec, Peter; Wang, Eddie C. Y.; Groh, Veronika; Spies, Thomas; McSharry, Brian P.; Aicheler, Rebecca J.; Stanton, Richard J.; Wilkinson, Gavin W. G.

    2007-01-01

    We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery. PMID:17374753

  2. Vector systems for prenatal gene therapy: principles of adenovirus design and production.

    PubMed

    Alba, Raul; Baker, Andrew H; Nicklin, Stuart A

    2012-01-01

    Adenoviruses have many attributes, which have made them one of the most widely investigated vectors for gene therapy applications. These include ease of genetic manipulation to produce replication-deficient vectors, ability to readily generate high titer stocks, efficiency of gene delivery into many cell types, and ability to encode large genetic inserts. Recent advances in adenoviral vector engineering have included the ability to genetically manipulate the tropism of the vector by engineering of the major capsid proteins, particularly fiber and hexon. Furthermore, simple replication-deficient adenoviral vectors deleted for expression of a single gene have been complemented by the development of systems in which the majority of adenoviral genes are deleted, generating sophisticated Ad vectors which can mediate sustained transgene expression following a single delivery. This chapter outlines methods for developing simple transgene over expressing Ad vectors and detailed strategies to engineer mutations into the major capsid proteins.

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

    PubMed Central

    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

  4. The role of capsid-endothelial interactions in the innate immune response to adenovirus vectors.

    PubMed

    Liu, Qiang; Zaiss, Anne K; Colarusso, Pina; Patel, Kamala; Haljan, Gregory; Wickham, Thomas J; Muruve, Daniel A

    2003-05-01

    Adenovirus (Ad) vectors can produce inflammatory responses at high doses. Intravenous administration of an Ad vector expressing green fluorescent protein (AdGFP) to naive mice induced a biphasic pattern of liver cytokine/chemokine gene expression over 7 days. Tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) genes were upregulated, with two distinct peaks of mRNA expression occurring at 6 hr and 5 days. The administration of transcription-defective AdGFP particles induced the early but not the late peak of chemokine/cytokine gene expression, confirming that Ad vector-induced inflammation is capsid dependent in the early phase and transcription dependent in the late phase. To determine the role of adenoviral capsid motifs in the early phase, capsid-modified Ad vectors were employed. The intravenous administration of the RGD-deleted Ad vector AdL.PB*, the fiber mutant AdL.F*, or the double mutant AdL.F*PB* induced similar levels of cytokine/chemokine expression compared with the wild-type vector AdLuc. Kupffer cell blockade significantly reduced liver TNF-alpha, MIP-2, and IP-10 gene expression and liver inflammation after the administration of AdL.PB* or AdL.F*PB*. Fluorescence microscopy of AdLuc- and AdL.PB*-transduced liver at 1 hr revealed localization of Ad vectors to liver sinusoids in Kupffer cell-depleted mice. AdL.PB* induced less E-selectin and VCAM-1 gene expression in liver, confirming reduced endothelial activation in mice receiving RGD-deleted Ad vectors. In vitro studies of endothelial cells demonstrated reduced transduction and endothelial activation by AdL.PB* compared with AdLuc. These results demonstrate that adenovirus capsid RGD motifs are required for efficient transduction and endothelial cell activation. Altering vector tropism represents a feasible strategy to modulate the innate response to Ad vectors in nontargeted tissues.

  5. Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28

    PubMed Central

    Kahl, Christoph A.; Bonnell, Jessica; Hiriyanna, Suja; Fultz, Megan; Nyberg-Hoffman, Cassandra; Chen, Ping; King, C. Richter; Gall, Jason G. D.

    2010-01-01

    Adenovirus vaccine vectors derived from rare human serotypes have been shown to be less potent than serotype 5 (Ad5) at inducing immune responses to encoded antigens. To identify highly immunogenic adenovirus vectors, we assessed pro-inflammatory cytokine expression, binding to the CD46 receptor, and immunogenicity. Species D adenoviruses uniquely suppressed pro-inflammatory cytokines and induced high levels of type I interferon. Thus, it was unexpected that a vector derived from a representative serotype, Ad28, induced significantly higher transgene-specific T-cell responses than an Ad35 vector. Prime-boost regimens with Ad28, Ad35, Ad14, or Ad5 significantly boosted T cell and antibody responses. The seroprevalence of Ad28 was confirmed to be <10% in the United States. Together, this shows that a rare human serotype-based vector can elicit strong immune responses, which was not predicted by in vitro results. PMID:20600496

  6. Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28.

    PubMed

    Kahl, Christoph A; Bonnell, Jessica; Hiriyanna, Suja; Fultz, Megan; Nyberg-Hoffman, Cassandra; Chen, Ping; King, C Richter; Gall, Jason G D

    2010-08-09

    Adenovirus vaccine vectors derived from rare human serotypes have been shown to be less potent than serotype 5 (Ad5) at inducing immune responses to encoded antigens. To identify highly immunogenic adenovirus vectors, we assessed pro-inflammatory cytokine expression, binding to the CD46 receptor, and immunogenicity. Species D adenoviruses uniquely suppressed pro-inflammatory cytokines and induced high levels of type I interferon. Thus, it was unexpected that a vector derived from a representative serotype, Ad28, induced significantly higher transgene-specific T cell responses than an Ad35 vector. Prime-boost regimens with Ad28, Ad35, Ad14, or Ad5 significantly boosted T cell and antibody responses. The seroprevalence of Ad28 was confirmed to be <10% in the United States. Together, this shows that a rare human serotype-based vector can elicit strong immune responses, which was not predicted by in vitro results.

  7. (13) C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

    PubMed

    Carinhas, Nuno; Koshkin, Alexey; Pais, Daniel A M; Alves, Paula M; Teixeira, Ana P

    2017-01-01

    Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-(13) C]glucose or [U-(13) C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary (13) C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

  8. Protection of Chickens against Avian Influenza with Non-Replicating Adenovirus-Vectored Vaccine

    PubMed Central

    Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Shi, Z.

    2009-01-01

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding an H7 AI hemagglutinin (AdChNY94.H7). Chickens vaccinated in ovo with an Ad vector encoding an AI H5 (AdTW68.H5) previously described, which were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Antibody responses to Ad vector in ovo vaccination follow a dose-response kinetic. The use of a synthetic AI H5 gene codon optimized to match the chicken cell tRNA pool was more potent than the cognate H5 gene. The use of Ad-vectored vaccines to increase resistance of chicken populations against multiple AI strains could reduce the risk of an avian-originating influenza pandemic in humans. PMID:18384919

  9. Evaluation of fiber-modified adenovirus vector-vaccine against foot-and-mouth diseaes in cattle

    USDA-ARS?s Scientific Manuscript database

    Novel vaccination approaches against foot-and-mouth-disease (FMD) include the use of a replication-defective human adenovirus type 5 vector (Ad5) that contains the capsid encoding regions of FMD virus (FMDV). An Ad5.A24 has proven effective as a vaccine against FMD in swine and cattle. However, ther...

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

    PubMed

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-01

    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.

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

    PubMed Central

    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 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. Methods 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. Results 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. Conclusions 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. PMID:24304565

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

    PubMed

    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.

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

    PubMed

    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.

  14. Adenovirus-derived vectors for prostate cancer gene therapy.

    PubMed

    de Vrij, Jeroen; Willemsen, Ralph A; Lindholm, Leif; Hoeben, Rob C; Bangma, Chris H; Barber, Chris; Behr, Jean-Paul; Briggs, Simon; Carlisle, Robert; Cheng, Wing-Shing; Dautzenberg, Iris J C; de Ridder, Corrina; Dzojic, Helena; Erbacher, Patrick; Essand, Magnus; Fisher, Kerry; Frazier, April; Georgopoulos, Lindsay J; Jennings, Ian; Kochanek, Stefan; Koppers-Lalic, Daniela; Kraaij, Robert; Kreppel, Florian; Magnusson, Maria; Maitland, Norman; Neuberg, Patrick; Nugent, Regina; Ogris, Manfred; Remy, Jean-Serge; Scaife, Michelle; Schenk-Braat, Ellen; Schooten, Erik; Seymour, Len; Slade, Michael; Szyjanowicz, Pio; Totterman, Thomas; Uil, Taco G; Ulbrich, Karel; van der Weel, Laura; van Weerden, Wytske; Wagner, Ernst; Zuber, Guy

    2010-07-01

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

  15. Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors

    PubMed Central

    Qiu, Qi; Xu, Zhili; Tian, Jie; Moitra, Rituparna; Gunti, Sreenivasulu; Notkins, Abner L.

    2014-01-01

    Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice. PMID:25552715

  16. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector

    PubMed Central

    Thomas, Michael A.; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

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

    PubMed

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K; Boling, Susan; Carroll, Jennifer L; Li, Xiao-Lin; Rogers, Donna L; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V; Curiel, David T; Mathis, J Michael

    2012-01-01

    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.

  18. Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

    PubMed Central

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K.; Boling, Susan; Carroll, Jennifer L.; Li, Xiao-Lin; Rogers, Donna L.; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V.; Curiel, David T.; Mathis, J. Michael

    2012-01-01

    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. PMID:23056548

  19. Adenovirus vector-induced immune responses in nonhuman primates: responses to prime boost regimens.

    PubMed

    Tatsis, Nia; Lasaro, Marcio O; Lin, Shih-Wen; Haut, Larissa H; Xiang, Zhi Q; Zhou, Dongming; Dimenna, Lauren; Li, Hua; Bian, Ang; Abdulla, Sarah; Li, Yan; Giles-Davis, Wynetta; Engram, Jessica; Ratcliffe, Sarah J; Silvestri, Guido; Ertl, Hildegund C; Betts, Michael R

    2009-05-15

    In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with preexisting neutralizing Abs against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here, we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee-derived Ad vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the chimpanzee-derived Ad vectors induced higher T and B cell responses than did repeated immunizations with the AdHu5 vector, especially in AdHu5-preexposed macaques.

  20. Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette†

    PubMed Central

    Massie, Bernard; Couture, France; Lamoureux, Linda; Mosser, Dick D.; Guilbault, Claire; Jolicoeur, Pierre; Bélanger, François; Langelier, Yves

    1998-01-01

    We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy. PMID:9499088

  1. Immobilization of 293 cells using porous support particles for adenovirus vector production

    PubMed Central

    Morishita, Naoya; Katsuda, Tomohisa; Kubo, Shuji; Gotoh, Akinobu

    2010-01-01

    Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 107 cells cm−3-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment. PMID:20140496

  2. [Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes].

    PubMed

    Sun, Zhi-hui; Han, Jie; Shao, Lei; Wang, Li-hong; Song, Jun-xian; Wei, Zhong-hai; Zheng, Liang-rong

    2010-05-01

    To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells. The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay. The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts. The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly

  3. Packaging capacity and stability of human adenovirus type 5 vectors.

    PubMed Central

    Bett, A J; Prevec, L; Graham, F L

    1993-01-01

    Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population. Images PMID:8371349

  4. Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

    PubMed Central

    Von Seggern, Dan J.; Huang, Shuang; Fleck, Shonna Kaye; Stevenson, Susan C.; Nemerow, Glen R.

    2000-01-01

    While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types. PMID:10590124

  5. Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine

    USDA-ARS?s Scientific Manuscript database

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FM...

  6. Syrian hamster tumor model to study oncolytic Ad5-based vectors.

    PubMed

    Dhar, Debanjan; Toth, Karoly; Wold, William S M

    2012-01-01

    Oncolytic (replicating) adenovirus (Ad) vectors are emerging as a promising form of a cancer therapy agent. There has been a need for an appropriate animal model to study oncolytic Ad since human Ad -replication is usually species specific. We have shown that Syrian (golden) hamsters are an appropriate animal model to study human Ad5-based vectors. Syrian hamsters are immunocompetent, and they allow human Ad5 replication in normal tissues as well as in Syrian hamster cancer cells. The development of the Syrian hamster as a model to study oncolytic Ad vectors has opened avenues to explore the role of host immune response and preexisting immunity in Ad vector efficacy and toxicity/biodistribution following Ad vector administration. Since most of the normal tissues in the Syrian hamster are permissive for human Ad5 replication, Ad vectors can be studied in the context of orthotopic cancer model developed in Syrian hamsters.

  7. Intracellular Signaling and Desmoglein 2 Shedding Triggered by Human Adenoviruses Ad3, Ad14, and Ad14P1

    PubMed Central

    Wang, Hongjie; Ducournau, Corinne; Saydaminova, Kamola; Richter, Maximilian; Yumul, Roma; Ho, Martin; Carter, Darrick; Zubieta, Chloé

    2015-01-01

    ABSTRACT We recently discovered that desmoglein 2 (DSG2) is a receptor for human adenovirus species B serotypes Ad3, Ad7, Ad11, and Ad14. Ad3 is considered to be a widely distributed human pathogen. Ad3 binding to DSG2 triggers the transient opening of epithelial junctions. Here, we further delineate the mechanism that leads to DSG2-mediated epithelial junction opening in cells exposed to Ad3 and recombinant Ad3 fiber proteins. We identified an Ad3 fiber knob-dependent pathway that involves the phosphorylation of mitogen-activated protein (MAP) kinases triggering the activation of the matrix-metalloproteinase ADAM17. ADAM17, in turn, cleaves the extracellular domain of DSG2 that links epithelial cells together. The shed DSG2 domain can be detected in cell culture supernatant and also in serum of mice with established human xenograft tumors. We then extended our studies to Ad14 and Ad14P1. Ad14 is an important research and clinical object because of the recent appearance of a new, more pathogenic strain (Ad14P1). In a human epithelial cancer xenograft model, Ad14P1 showed more efficient viral spread and oncolysis than Ad14. Here, we tested the hypothesis that a mutation in the Ad14P1 fiber knob could account for the differences between the two strains. While our X-ray crystallography studies suggested an altered three-dimensional (3D) structure of the Ad14P1 fiber knob in the F-G loop region, this did not significantly change the fiber knob affinity to DSG2 or the intracellular signaling and DSG2 shedding in epithelial cancer cells. IMPORTANCE A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 allows the virus not only to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus-triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber

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

    PubMed

    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.

  9. Toxicity associated with repeated administration of first-generation adenovirus vectors does not occur with a helper-dependent vector.

    PubMed Central

    O'Neal, W. K.; Zhou, H.; Morral, N.; Langston, C.; Parks, R. J.; Graham, F. L.; Kochanek, S.; Beaudet, A. L.

    2000-01-01

    BACKGROUND: Certain gene therapy protocols may require multiple administrations of vectors to achieve therapeutic benefit to the patient. This may be especially relevant for vectors such as adenoviral vectors that do not integrate into the host chromosome. Because immunocompetent animal models used for gene transfer studies develop neutralizing antibodies to adenoviral vectors after a single administration, little is known about how repeat administrations of vectors might affect transgene expression and vector toxicity. MATERIALS AND METHODS: We used mice deficient in the membrane spanning region of immunoglobulin (IgM), which do not develop antibodies, to evaluate the effect of repeated intravenous administration of first-generation and helper-dependent adenoviral vectors expressing human alpha 1-antitrypsin (hAAT). The duration and levels of transgene expression were evaluated after repeated administration of vectors. Toxicity was assessed by measuring the level of liver enzymes in the serum and the degrees of hepatocyte hypertrophy and proliferation. RESULTS: We found that previous administration of first-generation adenoviral vectors can alter the response to subsequent doses. These alterations included an increase in transgene expression early (within 1 and 3 days), followed by a rapid drop in expression by day 7. In addition, previous administrations of first-generation vectors led to an increase in toxicity of subsequent doses, as indicated by a rise in liver enzymes and an increase in hepatocyte proliferation. In contrast to first-generation vectors, use of the helper-dependent adenovirus vector, Ad-STK109, which contained no viral coding regions, did not lead to increased toxicity after multiple administrations. CONCLUSIONS: We conclude that the response of the host to adenoviral vectors can be altered after repeated administration, compared with the response after the initial vector dose. In addition, these experiments provide further evidence for the

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

    PubMed

    Szolajska, Ewa; Burmeister, Wim P; Zochowska, Monika; Nerlo, Barbara; Andreev, Igor; Schoehn, Guy; Andrieu, Jean-Pierre; Fender, Pascal; Naskalska, Antonina; Zubieta, Chloe; Cusack, Stephen; Chroboczek, Jadwiga

    2012-01-01

    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.

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

    PubMed Central

    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

    2015-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. PMID:26587311

  12. Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine

    USDA-ARS?s Scientific Manuscript database

    A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) sero-type O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa empty capsids. Swine inoculated with Ad5-O1Man developed an FMDV-specific neutralizing antibody response as compared to animals inoculated wi...

  13. Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells.

    PubMed

    Hosoya, Noriaki; Miura, Toshiyuki; Kawana-Tachikawa, Ai; Koibuchi, Tomohiko; Shioda, Tatsuo; Odawara, Takashi; Nakamura, Tetsuya; Kitamura, Yoshihiro; Kano, Munehide; Kato, Atsushi; Hasegawa, Mamoru; Nagai, Yoshiyuki; Iwamoto, Aikichi

    2008-03-01

    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients.

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

    PubMed Central

    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

  15. Improving gene transfer in human renal carcinoma cells: Utilization of adenovirus vectors containing chimeric type 5 and type 35 fiber proteins

    PubMed Central

    ACHARYA, BISHNU; TERAO, SHUJI; SUZUKI, TORU; NAOE, MICHIO; HAMADA, KATSUYUKI; MIZUGUCHI, HIROYUKI; GOTOH, AKINOBU

    2010-01-01

    The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally low due to the down-regulated expression of Coxsackie and adenovirus receptor (CAR) in target cells. By contrast, the infectivity of adenovirus serotype 35 vectors depends on the binding rate to CD46 receptor, independent of CAR. In this study, we examined whether an adenovirus vector containing chimeric type 5 and type 35 fiber proteins (Ad5/F35) increases transduction efficiency compared to Ad5 vector in human renal carcinoma cells in vitro. The expression of CAR was much lower in the human renal carcinoma cells than in control HEK293 cells. By contrast, the expression of CD46 was similar and perhaps at a higher level in the human renal carcinoma cells than in the HEK293 cells. The transduction efficacy of Ad5/F35 vector was dramatically higher compared to that of Ad5 in human renal carcinoma cells, and was correlated to the expression of CD46. Thus, Ad5/35 vector may be useful for the development of novel gene therapy approaches to renal cell carcinoma. PMID:22993573

  16. Optimization of adenovirus vectors for transduction in embryonic stem cells and induced pluripotent stem cells.

    PubMed

    Tashiro, Katsuhisa

    2011-01-01

      Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.

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

    PubMed Central

    Uusi-Kerttula, Hanni; Hulin-Curtis, Sarah; Davies, James; Parker, Alan L.

    2015-01-01

    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. PMID:26610547

  18. Characterization of an oncolytic adenovirus vector constructed to target the cMet receptor

    PubMed Central

    Sakr, Hany I; Coleman, David T; Cardelli, James A; Mathis, J Michael

    2015-01-01

    The cMet receptor is a homodimer with tyrosine kinase activity. Upon stimulation with its ligand, hepatocyte growth factor (HGF), the receptor mediates wide physiologic actions. The HGF-cMet signaling pathway is dysregulated in many cancers, which makes cMet an important target for novel therapeutic interventions. Oncolytic adenoviruses (Ads) have been used for the past three decades as a promising therapeutic approach for a wide array of neoplastic diseases. To date, achieving cancer-specific replication of oncolytic Ads has been accomplished by either viral genome deletions or by incorporating tumor selective promoters. To achieve novel specificity of oncolytic Ad infection of cancer cells that overexpress cMet, we inserted the HGF NK2 sequence, corresponding to a competitive antagonist of HGF binding to the cMet receptor, into the Ad serotype 5 (Ad5) fiber gene. The resulting vector, Ad5-pIX-RFP-FF/NK2, was rescued, amplified in HEK293 cells, and characterized. Binding specificity and viral infectivity were tested in various cancer cell lines that express varying levels of cMet and hCAR (the Ad5 receptor). We found that Ad5-pIX-RFP-FF/NK2 demonstrated binding specificity to the cMet receptor. In addition, there was enhanced viral infectivity and virus replication compared with a non-targeted Ad vector. Although NK2 weakly induces cMet receptor activation, our results showed no receptor phosphorylation in the context of an oncolytic Ad virus. In summary, these results suggest that an oncolytic Ad retargeted to the cMet receptor is a promising vector for developing a novel cancer therapeutic agent. PMID:26866014

  19. [Effects of recombinant adenovirus Ad-miR-29b2c on HGC-27 cell proliferation and migration].

    PubMed

    Luo, Ailing; Du, Chunyuan; Zhao, Xuemei; Liang, Jichao; Chen, Yong

    2017-07-25

    We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.

  20. Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

    PubMed

    Maeda, Mitsuko; Kida, Shinya; Hojo, Keiko; Eto, Yusuke; Gaob, Jian-Qing; Kurachi, Shinnosuke; Sekiguchi, Fumiko; Mizuguchi, Hiroyuki; Hayakawa, Takao; Mayumi, Tadanori; Nakagawa, Shinsaku; Kawasaki, Koichi

    2005-02-01

    The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

  1. Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

    PubMed

    Miura, Y; Yoshida, K; Nishimoto, T; Hatanaka, K; Ohnami, S; Asaka, M; Douglas, J T; Curiel, D T; Yoshida, T; Aoki, K

    2007-10-01

    Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

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

    PubMed

    Laakkonen, Johanna P; Engler, Tatjana; Romero, Ignacio A; Weksler, Babette; Couraud, Pierre-Olivier; Kreppel, Florian; Kochanek, Stefan

    2012-01-01

    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.

  3. [Producing recombinant adenovirus encoding green fluorescent protein (Ad-GFP) by suspension cultured HEK-293 N3S cells].

    PubMed

    Tian, Bo; Wu, Bin; Zhang, Qun-Wei; Bi, Jian-Jin; Wang, Lan; Zhu, Bao-Zhen; Geng, Yue; Wu, Zu-Ze

    2007-09-01

    Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

  4. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

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

    SciTech Connect

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

    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.

  6. Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene.

    PubMed

    Esandi, M C; van Someren, G D; Vincent, A J; van Bekkum, D W; Valerio, D; Bout, A; Noteboom, J L

    1997-04-01

    Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local

  7. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  8. Chimeric adenovirus type 5/35 vector encoding SIV gag and HIV env genes affords protective immunity against the simian/human immunodeficiency virus in monkeys.

    PubMed

    Someya, Kenji; Xin, Ke-Qin; Ami, Yasushi; Izumi, Yasuyuki; Mizuguchi, Hiroyuki; Ohta, Shinrai; Yamamoto, Naoki; Honda, Mitsuo; Okuda, Kenji

    2007-10-25

    Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.

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

    PubMed Central

    Crosby, Catherine M.; Barry, Michael A.

    2017-01-01

    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 immunodeficient mice, SC-Ad

  10. Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121.

    PubMed

    Lee, L Y; Patel, S R; Hackett, N R; Mack, C A; Polce, D R; El-Sawy, T; Hachamovitch, R; Zanzonico, P; Sanborn, T A; Parikh, M; Isom, O W; Crystal, R G; Rosengart, T K

    2000-01-01

    Adenovirus (Ad) vector-mediated gene therapy strategies have emerged as promising modalities for the "biological revascularization" of tissues. We hypothesized that direct intramyocardial, as opposed to intracoronary, administration of an Ad vector coding for the vascular endothelial growth factor 121 cDNA (Ad(GV)VEGF121.10) would provide highly focal Ad genome levels, and increases in VEGF, ideal for inducing localized therapeutic angiogenesis. Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial Ad(GV)VEGF121.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model. Intramyocardial delivery of Ad(GV)VEGF121.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial Ad(GV)VEGF121.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial Ad(GV)VEGF121.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration. These studies demonstrate that direct intramyocardial administration of Ad(GV)VEGF121.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human

  11. Enhanced adenovirus infection of melanoma cells by fiber-modification: incorporation of RGD peptide or Ad5/3 chimerism.

    PubMed

    Volk, Andrea L; Rivera, Angel A; Kanerva, Anna; Bauerschmitz, Gerd; Dmitriev, Igor; Nettelbeck, Dirk M; Curiel, David T

    2003-01-01

    The incidence of malignant melanoma has been increasing. Unfortunately, advanced melanomas are rarely curable with standard therapy; therefore, new forms of treatment such as gene therapy are needed. The success of gene delivery or oncolysis depends on the nature of the vector. Adenoviral vectors are advantageous for several reasons; however, they are dependent on CAR (coxsackie and adenovirus receptor) which is deficient or heterogeneously expressed on melanoma cells in situ. Correspondingly, transduction of freshly purified melanoma cells has been shown to be minimal or variable. In order to overcome this shortcoming, it is necessary to construct tropism modified adenoviral vectors. With this goal in mind, we generated two tropism modified vectors, Ad5lucRGD which has an RGD motif incorporated into the HI loop of the fiber knob and Ad5/3luc1 which contains the tail and shaft domain of the Ad5 fiber and the knob domain of the Ad3 fiber. Herein we demonstrate that Ad5/3luc1 infects CAR-negative primary melanoma cells 1128 times better than Ad5luc1 and 34 times better than Ad5lucRGD. Furthermore we show that Ad5/3luc1 and Ad5lucRGD infect via a CAR independent route by blocking the CAR receptor. In addition, we show that the infectivity of the cells correlates with the expression of CAR and Ad3 receptors determined by FACS analysis. Therefore, Ad5/3 is very attractive as a potential therapeutic vector for malignant melanoma.

  12. Adenovirus vectored vaccines against influenza a virus do not result in vaccine associated enhanced respiratory disease following heterologous challenge in contrast to whole inactivated virus vaccine

    USDA-ARS?s Scientific Manuscript database

    Heterologous influenza A virus (IAV) challenge following vaccination with an intramuscular (IM) whole inactivated vaccine (WIV) can result in vaccine-associated enhanced respiratory disease (VAERD). The objective of this study was to use an adenovirus (Ad5) vector vaccine platform that expressed IAV...

  13. Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes.

    PubMed

    Zhao, Chunxia; Crews, Charles Jefferson; Derdeyn, Cynthia A; Blackwell, Jerry L

    2009-09-01

    Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This

  14. Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector.

    PubMed

    Wising, Catharina; Magnusson, Maria; Ahlman, Karin; Lindholm, Leif; Lagergård, Teresa

    2010-02-01

    The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

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

    SciTech Connect

    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. . E-mail: dongj@genphar.com

    2006-09-30

    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{sup 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.

  16. Electrostatic Interactions between Complement Regulator CD46(SCR1-2) and Adenovirus Ad11/Ad21 Fiber Protein Knob

    PubMed Central

    Chen, Carl Z.; Gorham, Ronald D.; Gaieb, Zied; Morikis, Dimitrios

    2015-01-01

    Adenoviruses bind to a variety of human cells to cause infection. Both the B2 adenovirus 11 and B1 adenovirus 21 use protein knobs to bind to complement regulator CD46(SCR1-2) in order to gain entry into host cells. In each complex, the two proteins are highly negatively charged but bind to each other at an interface with oppositely charged surface patches. We computationally generated single-alanine mutants of charged residues in the complexes CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. We used electrostatic clustering and Poisson-Boltzmann free energy calculations to propose a hypothesis on the role of electrostatics in association. Our results delineate specific interfacial electrostatic interactions that are critical for association in both CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. These results will serve as a predictive tool in the selection of mutants with desired binding affinity in experimental mutagenesis studies. This study will also serve as a foundation for the design of inhibitors to treat adenovirus infections. PMID:26357573

  17. Electrostatic Interactions between Complement Regulator CD46(SCR1-2) and Adenovirus Ad11/Ad21 Fiber Protein Knob.

    PubMed

    Chen, Carl Z; Gorham, Ronald D; Gaieb, Zied; Morikis, Dimitrios

    2015-01-01

    Adenoviruses bind to a variety of human cells to cause infection. Both the B2 adenovirus 11 and B1 adenovirus 21 use protein knobs to bind to complement regulator CD46(SCR1-2) in order to gain entry into host cells. In each complex, the two proteins are highly negatively charged but bind to each other at an interface with oppositely charged surface patches. We computationally generated single-alanine mutants of charged residues in the complexes CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. We used electrostatic clustering and Poisson-Boltzmann free energy calculations to propose a hypothesis on the role of electrostatics in association. Our results delineate specific interfacial electrostatic interactions that are critical for association in both CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. These results will serve as a predictive tool in the selection of mutants with desired binding affinity in experimental mutagenesis studies. This study will also serve as a foundation for the design of inhibitors to treat adenovirus infections.

  18. Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle

    PubMed Central

    Medina, Gisselle N.; Montiel, Nestor; Diaz-San Segundo, Fayna; Sturza, Diego; Ramirez-Medina, Elizabeth; Grubman, Marvin J.

    2015-01-01

    Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4+ and CD8+ gamma interferon (IFN-γ)+ cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle. PMID:26607309

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

    PubMed Central

    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

  20. Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

    PubMed Central

    Torres, J M; Alonso, C; Ortega, A; Mittal, S; Graham, F; Enjuanes, L

    1996-01-01

    The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals

  1. Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

    PubMed Central

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

    2004-01-01

    Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These

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

    PubMed

    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. Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors.

    PubMed

    Ushitora, Masahiro; Sakurai, Fuminori; Yamaguchi, Tomoko; Nakamura, Shin-ichiro; Kondoh, Masuo; Yagi, Kiyohito; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2010-03-19

    Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury; however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase, which is an antioxidant enzyme scavenging H(2)O(2), resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model, CCl(4)-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing beta-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl(4)-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury. (c) 2009. Published by Elsevier B.V. All rights reserved.

  4. A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

    PubMed Central

    Parks, R J; Graham, F L

    1997-01-01

    Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors. PMID:9060698

  5. Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting.

    PubMed

    Garas, M N; Tillib, S V; Zubkova, O V; Rogozhin, V N; Ivanova, T I; Vasilev, L A; Logunov, D Yu; Shmarov, M M; Tutykhina, I L; Esmagambetov, I B; Gribova, I Yu; Bandelyuk, A S; Naroditsky, B S; Gintsburg, A L

    2014-04-01

    Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.

  6. Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting

    PubMed Central

    Garas, M. N.; Tillib, S. V.; Zubkova, O. V.; Rogozhin, V. N.; Ivanova, T. I.; Vasilev, L. A.; Logunov, D. Yu.; Shmarov, M. M.; Tutykhina, I. L.; Esmagambetov, I. B.; Gribova, I. Yu.; Bandelyuk, A. S.; Naroditsky, B. S.; Gintsburg, A. L.

    2014-01-01

    Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to “nanoantibody–modified RPAN” binding. PMID:25093116

  7. Inhibition of NF-κB Activation in Combination with Bcl-2 Expression Allows for Persistence of First-Generation Adenovirus Vectors in the Mouse Liver

    PubMed Central

    Lieber, André; He, Chen-Yi; Meuse, Leonard; Himeda, Charis; Wilson, Christopher; Kay, Mark A.

    1998-01-01

    NF-κB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-κB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IκBα supersuppressor (Ad.IκBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-κB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IκBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 × C3H mice. In the absence of Bcl-2, IκBM expression in mouse livers significantly reduced NF-κB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IκBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-κB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-κB protection was inactivated by IκBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence. PMID:9765474

  8. Truncated Active Human Matrix Metalloproteinase-8 Delivered by a Chimeric Adenovirus-Hepatitis B Virus Vector Ameliorates Rat Liver Cirrhosis

    PubMed Central

    Wang, Zihua; Li, Dong; Kang, Fubiao; Li, Haijun; Li, Baosheng; Cao, Zhichen; Nassal, Michael; Sun, Dianxing

    2013-01-01

    Background Liver cirrhosis is a potentially life-threatening disease caused by progressive displacement of functional hepatocytes by fibrous tissue. The underlying fibrosis is often driven by chronic infection with hepatitis B virus (HBV). Matrix metalloproteinases including MMP-8 are crucial for excess collagen degradation. In a rat model of liver cirrhosis, MMP-8 delivery by an adenovirus (Ad) vector achieved significant amelioration of fibrosis but application of Ad vectors in humans is subject to various issues, including a lack of intrinsic liver specificity. Methods HBV is highly liver-specific and its principal suitability as liver-specific gene transfer vector is established. HBV vectors have a limited insertion capacity and are replication-defective. Conversely, in an HBV infected cell vector replication may be rescued in trans by the resident virus, allowing conditional vector amplification and spreading. Capitalizing on a resident pathogen to help in its elimination and/or in treating its pathogenic consequences would provide a novel strategy. However, resident HBV may also reduce susceptibility to HBV vector superinfection. Thus a size-compatible truncated MMP-8 (tMMP8) gene was cloned into an HBV vector which was then used to generate a chimeric Ad-HBV shuttle vector that is not subject to superinfection exclusion. Rats with thioacetamide-induced liver cirrhosis were injected with the chimera to evaluate therapeutic efficacy. Results Our data demonstrate that infectious HBV vector particles can be obtained via trans-complementation by wild-type virus, and that the tMMP8 HBV vector can efficiently be shuttled by an Ad vector into cirrhotic rat livers. There it exerted a comparable beneficial effect on fibrosis and hepatocyte proliferation markers as a conventional full-length MMP-8Ad vector. Conclusions Though the rat cirrhosis model does not allow assessing in vivo HBV vector amplification these results advocate the further development of Ad

  9. Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

    PubMed Central

    Kaner, Robert J.; Santiago, Francisco; Rahaghi, Franck; Michaels, Elizabeth; Moore, John P.; Crystal, Ronald G.

    2010-01-01

    Heterologous viruses may transactivate or suppress human immunodeficiency virus (HIV)–1 replication. An adenovirus type 5 gene transfer vector (Ad5) HIV-1 vaccine was recently evaluated in a clinical trial, without efficacy. In this context, it is relevant to ask what effect Ad vectors have on HIV-1 replication, particularly in cells that are part of the innate immune system. Infection of HIV-1–infected human alveolar macrophages (AMs) obtained from HIV-1+ individuals with an Ad vector containing no transgene (AdNull) resulted in dose-responsive inhibition of endogenous HIV-1 replication. HIV-1 replication in normal AMs infected with HIV-1 in vitro was inhibited by AdNull with a similar dose response. Ad reduced AM HIV-1 replication up to 14 days after HIV-1 infection. Fully HIV-1–infected AMs were treated with 3′-azido-3′-deoxythymidine, after which Ad infection still inhibited HIV-1 replication, suggesting a postentry step was affected. Substantial HIV-1 DNA was still produced after Ad infection, as quantified by TaqMan real-time PCR, suggesting that the replication block occurred after reverse transcription. AdNull blocked HIV-1 long terminal repeat (LTR) transcription, as assessed by an vesicular stomatitis virus G protein pseudotyped HIV-1 LTR luciferase construct. The formation of HIV-1 DNA integrated into the host chromosome was not inhibited by Ad, as quantified by a two-step TaqMan real-time PCR assay, implying a postintegration block to HIV-1 replication. These data indicate that Ad vectors are inhibitory to HIV-1 replication in human AMs based, in part, on their ability to inhibit LTR-driven transcription. PMID:19805482

  10. Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity

    PubMed Central

    Anghelina, Daniela; Lam, Eric

    2016-01-01

    ABSTRACT Infection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs) in vitro. We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS−/− and STING−/− mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections. IMPORTANCE The detection of viral infection by host sentinel immune cells

  11. [Influence of c-kit RNA interference mediated by AdMax adenovirus upon gastrointestinal stromal tumor in nude mice].

    PubMed

    Wang, Tian-bao; Shi, Han-ping; Huang, Wen-sheng; Lin, Wei-hao; Dong, Wen-guang

    2011-03-01

    To investigate a novel therapeutic regiment for gastrointestinal stromal tumor (GIST) based on c-kit RNA interference (RNAi) under the mediation of AdMax adenovirus. c-kit shRNA, whose lateral sides were decorated with restriction endonuclease sequences, was designed. The joining of c-kit shRNA and PDC316-EGFP-U6 was catalyzed by T4 DNA ligase to construct PDC316-EGFP-U6-C-KIT. Homologous recombination of AdEGFP-U6-C-KIT was performed with AdMax system. Heterotopic transplantation of GIST in nude mice was established. AdEGFP-U6-C-KIT was intratumorally injected in experimental group while blank admax adenovirus AdEGFP-U6 in control group. The volume, inhibition ratio of tumor and CD117 expression of graft tumor were compared between test and control groups. The length of c-kit shRNA was around 50 bp in agarose electrophoresis. Gene sequencing revealed the designed c-kit RNAi sequence in PDC316-EGFP-U6-C-KIT. After transfection with AdEGFP-U6-C-KIT, 293 cells presented green fluorescence. The physical and infective titer of AdEGFP-U6-C-KIT was 5 × 10(11)vp/ml and 5.67 × 10(7) pfu/ml respectively. At the end of test, the mean volume of graft tumor was significantly smaller in test group than in control group [(75 ± 23) vs (989 ± 31) mm(3), P = 0.000]. The inhibition ratio of tumor was 59.6% in test group. Two cases (20%) in test group and 10 (100%) in control group had a positive expression of CD117 (P = 0.001). c-kit RNAi mediated by Admax vector system can inhibit effectively the expression of c-kit gene and the growth of GIST in nude mice.

  12. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  13. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    NASA Astrophysics Data System (ADS)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  14. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    PubMed Central

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí

    2015-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

  15. Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters.

    PubMed

    Schaar, Katrin; Geisler, Anja; Kraus, Milena; Pinkert, Sandra; Pryshliak, Markian; Spencer, Jacqueline F; Tollefson, Ann E; Ying, Baoling; Kurreck, Jens; Wold, William S; Klopfleisch, Robert; Toth, Karoly; Fechner, Henry

    2017-09-15

    Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

    PubMed Central

    Benlahrech, Adel; Harris, Julian; Meiser, Andrea; Papagatsias, Timos; Hornig, Julia; Hayes, Peter; Lieber, Andre; Athanasopoulos, Takis; Bachy, Veronique; Csomor, Eszter; Daniels, Rod; Fisher, Kerry; Gotch, Frances; Seymour, Len; Logan, Karen; Barbagallo, Romina; Klavinskis, Linda; Dickson, George; Patterson, Steven

    2009-01-01

    In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition. PMID:19918060

  17. Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

    PubMed

    Doronin, K K; Zakharchuk, A N; Grinenko, N F; Yurov, G K; Krougliak, V A; Naroditsky, B S

    1993-04-30

    A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.

  18. Suicide gene therapy using adenovirus vector for human oral squamous carcinoma cell line in vitro.

    PubMed

    Yamamoto, Noriyuki; Hayashi, Yasushi; Kagami, Hideaki; Fukui, Takafumi; Fukuhara, Hirokazu; Tohnai, Iwai; Ueda, Minoru; Mizuno, Masaaki; Yoshida, Jun

    2005-06-01

    Recently, suicide gene therapy using the herpes simplex virus thymidine kinase (HSVtk) gene followed by ganciclovir (GCV) administration was evaluated for the treatment of cancer. The purpose of this study was to investigate the effectiveness of suicide gene therapy using the replication-deficient recombinant adenovirus vector for human oral squamous carcinoma cell lines. To evaluate transduction efficiency, each cell line was transduced in vitro with an adenovirus vector containing the beta-galactosidase gene. By 24 hours after transduction, nearly 100% of the cells were transduced at a multiplicity of infection (MOI) of 10, and from 30 to 10% at an MOI of 1. Next, each cell line was transduced with an adenovirus vector containing the HSVtk gene, and a subsequent administration of GCV for the assessment of suicide gene therapy. A subsequent administration of GCV resulted in complete tumor cell death. In addition, we conducted a morphological analysis of that cell death using video-enhanced contrast differential interference contrast microscopy, and we observed that it included both apoptosis and necrosis after HSVtk gene and GCV treatment. These results suggest that adenovirus-mediated suicide gene therapy induced remarkable cytotoxicity with a bystander effect in human oral squamous cell carcinoma thus suggesting an effective treatment strategy for that tumor.

  19. Vaccination of rabbits with an adenovirus vector expressing the papillomavirus E2 protein leads to clearance of papillomas and infection.

    PubMed

    Brandsma, Janet L; Shlyankevich, Mark; Zhang, Lixin; Slade, Martin D; Goodwin, Edward C; Peh, Woei; Deisseroth, Albert B

    2004-01-01

    Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.

  20. Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus

    PubMed Central

    Xiao, Xiao; Li, Juan; Samulski, Richard Jude

    1998-01-01

    Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236–5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 105 viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use. PMID:9499080

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

    SciTech Connect

    Campos, Samuel K.; Barry, Michael A. . E-mail: mab@bcm.edu

    2006-06-05

    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.

  2. Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    PubMed Central

    Suzuki, Mariko; Kondo, Saki; Yamasaki, Manabu; Matsuda, Norie; Nomoto, Akio; Suzuki, Tetsuro; Saito, Izumu; Kanegae, Yumi

    2017-01-01

    The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely. PMID:28157182

  3. Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom.

    PubMed

    Zeng, Zhiwei; Zhang, Jing; Jing, Shuping; Cheng, Zetao; Bofill-Mas, Silvia; Maluquer de Motes, Carlos; Hundesa, Ayalkibet; Girones, Rosina; Seto, Donald; Zhang, Qiwei

    2016-11-03

    The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

  4. Adenovirus capsid-display of the retro-oriented human complement inhibitor DAF reduces Ad vector–triggered immune responses in vitro and in vivo

    PubMed Central

    Seregin, Sergey S.; Aldhamen, Yasser A.; Appledorn, Daniel M.; Hartman, Zachary C.; Schuldt, Nathaniel J.; Scott, Jeannine; Godbehere, Sarah; Jiang, Haixiang; Frank, Michael M.

    2010-01-01

    Adenovirus (Ad) vectors are widely used in human clinical trials. However, at higher dosages, Ad vector–triggered innate toxicities remain a major obstacle to many applications. Ad interactions with the complement system significantly contribute to innate immune responses in several models of Ad-mediated gene transfer. We constructed a novel class of Ad vectors, genetically engineered to “capsid-display” native and retro-oriented versions of the human complement inhibitor decay-accelerating factor (DAF), as a fusion protein from the C-terminus of the Ad capsid protein IX. In contrast to conventional Ad vectors, DAF-displaying Ads dramatically minimized complement activation in vitro and complement-dependent immune responses in vivo. DAF-displaying Ads did not trigger thrombocytopenia, minimized endothelial cell activation, and had diminished inductions of proinflammatory cytokine and chemokine responses. The retro-oriented display of DAF facilitated the greatest improvements in vivo, with diminished activation of innate immune cells, such as dendritic and natural killer cells. In conclusion, Ad vectors can capsid-display proteins in a manner that not only retains the functionality of the displayed proteins but also potentially can be harnessed to improve the efficacy of this important gene transfer platform for numerous gene transfer applications. PMID:20511542

  5. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

    PubMed Central

    Mittereder, N; March, K L; Trapnell, B C

    1996-01-01

    Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in

  6. Tropism modification of adenovirus vectors by peptide ligand insertion into various positions of the adenovirus serotype 41 short-fiber knob domain.

    PubMed

    Hesse, Andrea; Kosmides, Daniela; Kontermann, Roland E; Nettelbeck, Dirk M

    2007-03-01

    Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

  7. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    PubMed Central

    Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.

    2013-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:23383334

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

    DTIC Science & Technology

    2006-06-01

    Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs Nancy J. Sullivan 1...Shedlock DJ, Xu L, et al. (2006) Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified...should be addressed. E-mail: gnabel@nih.gov [ These authors contributed equally to this work. A B S T R A C T Background Ebola virus causes a hemorrhagic

  9. In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

    PubMed Central

    Richter, Maximilian; Saydaminova, Kamola; Yumul, Roma; Krishnan, Rohini; Liu, Jing; Nagy, Eniko-Eva; Singh, Manvendra; Izsvák, Zsuzsanna; Cattaneo, Roberto; Uckert, Wolfgang; Palmer, Donna; Ng, Philip; Haworth, Kevin G.; Kiem, Hans-Peter; Ehrhardt, Anja; Papayannopoulou, Thalia

    2016-01-01

    Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin−Sca1+Kit− cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. PMID:27554082

  10. Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle.

    PubMed

    Medina, Gisselle N; Montiel, Nestor; Diaz-San Segundo, Fayna; Sturza, Diego; Ramirez-Medina, Elizabeth; Grubman, Marvin J; de los Santos, Teresa

    2015-11-25

    Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4(+) and CD8(+) gamma interferon (IFN-γ)(+) cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. A Genetically Engineered Adenovirus Vector Targeted to CD40 Mediates Transduction of Canine Dendritic Cells and Promotes Antigen-Specific Immune Responses In Vivo

    PubMed Central

    Thacker, Erin E.; Nakayama, Masaharu; Smith, Bruce F.; Bird, R. Curtis; Muminova, Zhanat; Strong, Theresa; Timares, Laura; Korokhov, Nikolay; O'Neill, Ann Marie; de Gruijl, Tanja D.; Glasgow, Joel N.; Tani, Kenzaburo; Curiel, David T.

    2009-01-01

    Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy. PMID:19786146

  12. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine.

    PubMed

    Pandey, Aseem; Singh, Neetu; Vemula, Sai V; Couëtil, Laurent; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2012-01-01

    The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

  13. Vasculature-Specific Adenovirus Vectors for Gene Therapy of Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    translation and, thus, never become available to the disulfide isomerases, the ER-localized enzymes that facilitate the formation of the disulfide bonds...duplexes and cloned into Bael-cut expression vectors. Upon transformation of Ecoli , colonies containing recombinant plasmids were identified by PCR...R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-3. 6

  14. Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors

    PubMed Central

    Yan, Jingyi; Dong, Jianing; Wu, Jiaxin; Zhu, Rui; Wang, Zhen; Wang, Baoming; Wang, Lizheng; Wang, Zixuan; Zhang, Haihong; Wu, Hui; Yu, Bin; Kong, Wei; Yu, Xianghui

    2016-01-01

    The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors. PMID:26934960

  15. Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors.

    PubMed

    Yan, Jingyi; Dong, Jianing; Wu, Jiaxin; Zhu, Rui; Wang, Zhen; Wang, Baoming; Wang, Lizheng; Wang, Zixuan; Zhang, Haihong; Wu, Hui; Yu, Bin; Kong, Wei; Yu, Xianghui

    2016-03-03

    The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.

  16. Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine.

    PubMed

    Lokhandwala, Shehnaz; Waghela, Suryakant D; Bray, Jocelyn; Sangewar, Neha; Charendoff, Chloe; Martin, Cameron L; Hassan, Wisam S; Koynarski, Tsvetoslav; Gabbert, Lindsay; Burrage, Thomas G; Brake, David; Neilan, John; Mwangi, Waithaka

    2017-01-01

    African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.

  17. Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine

    PubMed Central

    Waghela, Suryakant D.; Bray, Jocelyn; Sangewar, Neha; Charendoff, Chloe; Martin, Cameron L.; Hassan, Wisam S.; Koynarski, Tsvetoslav; Gabbert, Lindsay; Burrage, Thomas G.; Brake, David; Neilan, John; Mwangi, Waithaka

    2017-01-01

    African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy. PMID:28481911

  18. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  19. Adenovirus as a gene therapy vector for hematopoietic cells.

    PubMed

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M

    2000-06-01

    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

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

    PubMed

    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.

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

    PubMed

    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.

  2. [Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4].

    PubMed

    Zhao, Jing; Xu, Guang-xian; Jia, Wei; Dong, Hui; Zhang, Yi-lin; Zhao, Zhi-jun; Wei, Jun

    2012-02-01

    To construct the recombined adenovirus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR- 21 with its target gene TLR4. Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  3. New Conditionally Replicating Adenovirus Vectors for Breast Cancer Therapy

    DTIC Science & Technology

    2009-09-01

    Baculovirus Construct Description of Mutation I664V Ad5 Pol – pAcgp67A Protein Production in SF9 insect cells I664M Ad5 Pol – pAcgp67A “” I664S...out in SF9 insect cells (Figure 3). Figure 3: Expression of Ad pol proteins in insect cells LEGEND: 3µg of recombinant baculovirus DNA was...transfected with Cellfectin (Invitrogen) in SF9 insect cells. Three days later the recombinant baculoviruses were collected and used for viral

  4. Phylogenetic Analysis and Structural Predictions of Human Adenovirus Penton Proteins as a Basis for Tissue-Specific Adenovirus Vector Design▿

    PubMed Central

    Madisch, Ijad; Hofmayer, Soeren; Moritz, Christian; Grintzalis, Alexander; Hainmueller, Jens; Pring-Akerblom, Patricia; Heim, Albert

    2007-01-01

    The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17′H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors. PMID:17522221

  5. Vector sequences are not detected in tumor tissue from research subjects with ornithine transcarbamylase deficiency who previously received adenovirus gene transfer.

    PubMed

    Zhong, Li; Li, Shaoyong; Li, Mengxin; Xie, Jun; Zhang, Yu; Lee, Brendan; Batshaw, Mark L; Wilson, James M; Gao, Guangping

    2013-09-01

    A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.

  6. The Ad5 [E1-, E2b-]-based vector: a new and versatile gene delivery platform

    NASA Astrophysics Data System (ADS)

    Jones, Frank R.; Gabitzsch, Elizabeth S.; Balint, Joseph P.

    2015-05-01

    Based upon advances in gene sequencing and construction, it is now possible to identify specific genes or sequences thereof for gene delivery applications. Recombinant adenovirus serotype-5 (Ad5) viral vectors have been utilized in the settings of gene therapy, vaccination, and immunotherapy but have encountered clinical challenges because they are recognized as foreign entities to the host. This recognition leads to an immunologic clearance of the vector that contains the inserted gene of interest and prevents effective immunization(s). We have reported on a new Ad5-based viral vector technology that can be utilized as an immunization modality to induce immune responses even in the presence of Ad5 vector immunity. We have reported successful immunization and immunotherapy results to infectious diseases and cancers. This improved recombinant viral platform (Ad5 [E1-, E2b-]) can now be utilized in the development of multiple vaccines and immunotherapies.

  7. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  8. Adenovirus Specific Pre-Immunity Induced by Natural Route of Infection Does Not Impair Transduction by Adenoviral Vaccine Vectors in Mice

    PubMed Central

    de Andrade Pereira, Bruna; E. Maduro Bouillet, Leoneide; Dorigo, Natalia A.; Fraefel, Cornel; Bruna-Romero, Oscar

    2015-01-01

    Recombinant human adenovirus serotype 5 (HAd5V) vectors are gold standards of T-cell immunogenicity as they efficiently induce also humoral responses to exogenous antigens, in particular when used in prime-boost protocols. Some investigators have shown that pre-existing immunity to adenoviruses interferes with transduction by adenoviral vectors, but the actual extent of this interference is not known since it has been mostly studied in mice using unnatural routes of infection and virus doses. Here we studied the effects of HAd5V-specific immune responses induced by intranasal infection on the transduction efficiency of recombinant adenovirus vectors. Of interest, when HAd5V immunity was induced in mice by the natural respiratory route, the pre-existing immunity against HAd5V did not significantly interfere with the B and T-cell immune responses against the transgene products induced after a prime/boost inoculation protocol with a recombinant HAd5V-vector, as measured by ELISA and in vivo cytotoxic T-cell assays, respectively. We also correlated the levels of HAd5V-specific neutralizing antibodies (Ad5NAbs) induced in mice with the levels of Ad5NAb titers found in humans. The data indicate that approximately 60% of the human serum samples tested displayed Ad5NAb levels that could be overcome with a prime-boost vaccination protocol. These results suggest that recombinant HAd5V vectors are potentially useful for prime-boost vaccination strategies, at least when pre-existing immunity against HAd5V is at low or medium levels. PMID:26679149

  9. Adenovirus Specific Pre-Immunity Induced by Natural Route of Infection Does Not Impair Transduction by Adenoviral Vaccine Vectors in Mice.

    PubMed

    de Andrade Pereira, Bruna; Maduro Bouillet, Leoneide E; Dorigo, Natalia A; Fraefel, Cornel; Bruna-Romero, Oscar

    2015-01-01

    Recombinant human adenovirus serotype 5 (HAd5V) vectors are gold standards of T-cell immunogenicity as they efficiently induce also humoral responses to exogenous antigens, in particular when used in prime-boost protocols. Some investigators have shown that pre-existing immunity to adenoviruses interferes with transduction by adenoviral vectors, but the actual extent of this interference is not known since it has been mostly studied in mice using unnatural routes of infection and virus doses. Here we studied the effects of HAd5V-specific immune responses induced by intranasal infection on the transduction efficiency of recombinant adenovirus vectors. Of interest, when HAd5V immunity was induced in mice by the natural respiratory route, the pre-existing immunity against HAd5V did not significantly interfere with the B and T-cell immune responses against the transgene products induced after a prime/boost inoculation protocol with a recombinant HAd5V-vector, as measured by ELISA and in vivo cytotoxic T-cell assays, respectively. We also correlated the levels of HAd5V-specific neutralizing antibodies (Ad5NAbs) induced in mice with the levels of Ad5NAb titers found in humans. The data indicate that approximately 60% of the human serum samples tested displayed Ad5NAb levels that could be overcome with a prime-boost vaccination protocol. These results suggest that recombinant HAd5V vectors are potentially useful for prime-boost vaccination strategies, at least when pre-existing immunity against HAd5V is at low or medium levels.

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

    PubMed Central

    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

  11. Novel cocaine vaccine linked to a disrupted adenovirus gene transfer vector blocks cocaine psychostimulant and reinforcing effects.

    PubMed

    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

    2012-04-01

    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 (3)H-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 (>10(5)) 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.

  12. [Effect of recombinant adenovirus Ad-DT-A in targeted therapy for malignant cancer cell lines with loss of IGF2 imprinting].

    PubMed

    Pan, Yu-qin; He, Bang-shun; Zhu, Chan; Qu, Li-li; Xu, Xiong-fei; Wang, Shu-kui

    2011-11-01

    To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors. The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors. The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%. The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of

  13. Adenovirus serotype 5 vaccine vectors trigger IL-27-dependent inhibitory CD4+ T cell responses that impair CD8+ T cell function

    PubMed Central

    Larocca, Rafael A.; Provine, Nicholas M.; Aid, Malika; Iampietro, M. Justin; Borducchi, Erica N.; Badamchi-Zadeh, Alexander; Abbink, Peter; Ng’ang’a, David; Bricault, Christine A.; Blass, Eryn; Penaloza-MacMaster, Pablo; Stephenson, Kathryn E.; Barouch, Dan H.

    2017-01-01

    Adenovirus serotype 5 (Ad5) vaccine vectors elicit robust CD8+ T cell responses, but these responses typically exhibit a partially exhausted phenotype. However, the immunologic mechanism by which Ad5 vectors induce dysfunctional CD8+ T cells has not previously been elucidated. Here we demonstrate that, following immunization of B6 mice, Ad5 vectors elicit antigen-specific IL-10+CD4+ T cells with a distinct transcriptional profile in a dose-dependent fashion. In rhesus monkeys, we similarly observed upregulated expression of IL-10 and PD-1 by CD4+ T cells following Ad5 vaccination. These cells markedly suppressed vaccine-elicited CD8+ T cell responses in vivo and IL-10 blockade increased the frequency and functionality of antigen-specific CD8+ T cells as well as improved protective efficacy against challenge with recombinant Listeria monocytogenes. Moreover, induction of these inhibitory IL-10+CD4+ T cells correlated with IL-27 expression and IL-27 blockade substantially improved CD4+ T cell functionality. These data highlight a role for IL-27 in the induction of inhibitory IL-10+CD4+ T cells, which suppress CD8+ T cell magnitude and function following Ad5 vector immunization. A deeper understanding of the cytokine networks and transcriptional profiles induced by vaccine vectors should lead to strategies to improve the immunogenicity and protective efficacy of viral vector-based vaccines. PMID:28239679

  14. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    PubMed

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian

    2013-02-01

    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (P<0.01). There was a significant difference in the expression level of BMP2 mRNA between group A and C (P<0.05) and this was markedly higher than that in groups B, D and E (P<0.01). The protein expression level of HIF1α in group A and B was markedly higher than that in groups C, D and E (P<0.01). The protein expression level of BMP2 in group A and C was markedly higher than that in groups B, D and E (P<0.01). The human BMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  15. Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors.

    PubMed

    Mitchell, M; Jerebtsova, M; Batshaw, M L; Newman, K; Ye, X

    2000-12-01

    We have developed a micro-injection technique to deliver recombinant adenovirus and AAV to mouse fetuses at day 15 after conception. Several routes of delivery, including injections to the amniotic fluid, the front limb, the placenta, the liver, and the retro-orbital venus plexus, were tested using an E1-deleted recombinant adenovirus (Ad.CBlacZ) or a recombinant adeno-associated virus (AAV.CMVlacZ) carrying a beta-galactosidase (lacZ) gene. Injection of Ad.CBlacZ into the amniotic cavity led to transgene expression in the skin and in the digestive tract of the fetuses. Injection of Ad.CBlacZ in the front limb resulted in LacZ expression in all major muscle groups around the injection site and at low levels in the liver. The other three routes of delivery, ie intra-placental, intra-hepatic and retro-orbital injections of Ad.CBlacZ, all led to lacZ expression predominantly in the liver. Further studies revealed a maximal tolerant dose (defined as the highest viral dose with < or =20% mortality in the injected fetuses) of 1 x 10(9) particles per fetus for intra- hepatic injections, 3 x 10(9) particles per fetus for intra-placental injection, 1 x 1010 particles per fetus for retro-orbital and intra-amniotic injections, and 2 x 10(10) particle per fetus for intra-muscular injection. The adenovirus-mediated lacZ expression in liver and muscle persisted for at least 6 weeks. Intra-muscular injection of AAV.CMVlacZ also resulted in lacZ expression in the muscle up to 3 months after birth with no indication of cellular immune response at the injection site. Taken together, our results demonstrated that prolonged transgene expression can be achieved by in utero gene transfer using either adenoviral or AAV vectors. The distribution of virus-mediated gene transfer appeared to determined mostly by the route of viral administration.

  16. Development and Pre-Clinical Evaluation of a Novel Prostate-Restricted Replication Competent Adenovirus-Ad-IU-1

    DTIC Science & Technology

    2005-05-01

    was performed using monoclonal antibodies against Ad5E1a or polyclonal HSV1 -TK antiserum. Ad5E1a and HSV1 -TK proteins expression were detected in...which are prominently expressed in androgen independent (AI) prostate cancers. The goal of this research is to develop a novel therapeutic agent, Ad...IU-1, using PSES to control the replication of adenovirus and the expression of a therapeutic gene, herpes simplex thymidine kinase (TK). AD-IU-1

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

    PubMed

    Simão, D; Pinto, C; Fernandes, P; Peddie, C J; Piersanti, S; Collinson, L M; Salinas, S; Saggio, I; Schiavo, G; Kremer, E J; Brito, C; Alves, P M

    2016-01-01

    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 preclinical tests. For clinical translation, in-depth preclinical 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, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward 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.

  18. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

    PubMed Central

    Gorziglia, M I; Kadan, M J; Yei, S; Lim, J; Lee, G M; Luthra, R; Trapnell, B C

    1996-01-01

    A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy. PMID:8648763

  19. Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine.

    PubMed

    Diaz-San Segundo, Fayna; Dias, Camila C; Moraes, Mauro P; Weiss, Marcelo; Perez-Martin, Eva; Salazar, Andres M; Grubman, Marvin J; de los Santos, Teresa

    2014-11-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FMDV challenge by 7 days post-vaccination. However, since relatively large amounts of Ad5-CI-A24-2B are required to induce protection this strategy could be costly for livestock production. Poly ICLC is a synthetic double stranded RNA that activates multiple innate and adaptive immune pathways. In this study, we have tested for the first time, the adjuvant effect of poly ICLC in combination with Ad5-CI-A24-2B in swine. We found that the combination resulted in a reduction of the vaccine protective dose by 80-fold. Interestingly, the lowest dose of Ad5-CI-A24-2B plus 1mg of poly ICLC protected animals against challenge even in the absence of detectable FMDV-specific neutralizing antibodies at the time of challenge.

  20. Functional characterization of a PEI-CyD-FA-coated adenovirus as delivery vector for gene therapy.

    PubMed

    Yao, Hong; Chen, Shih-Chi; Shen, Zan; Huang, Yun-Chao; Zhu, Xiao; Wang, Xiao-mei; Jiang, Wenqi; Wang, Zi-Feng; Bian, Xiu-Wu; Ling, Eng-Ang; Kung, Hsiang-fu; Lin, Marie C

    2013-01-01

    The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy.

  1. Mucosal Trafficking of Vector-Specific CD4+ T Lymphocytes following Vaccination of Rhesus Monkeys with Adenovirus Serotype 5▿ †

    PubMed Central

    Masek-Hammerman, Katherine; Li, Hualin; Liu, Jinyan; Abbink, Peter; La Porte, Annalena; O'Brien, Kara L.; Whitney, James B.; Carville, Angela; Mansfield, Keith G.; Barouch, Dan H.

    2010-01-01

    Post hoc analysis of the phase 2b Step study evaluating a recombinant adenovirus serotype 5 (rAd5)-based HIV-1 vaccine candidate suggested a potential increased risk of HIV-1 acquisition in subjects who were baseline Ad5 seropositive and uncircumcised. These concerns had a profound impact on the HIV-1 vaccine development field, although the mechanism underlying this observation remains unknown. It has been hypothesized that rAd5 vaccination of baseline Ad5-seropositive individuals may have resulted in anamnestic, vector-specific CD4+ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Here we show that Ad5-specific CD4+ T lymphocyte responses at mucosal sites following rAd5-Gag/Pol/Nef vaccination were comparable in rhesus monkeys with and without baseline Ad5 immunity. Moreover, the total cellular inflammatory infiltrates and the CD3+, CD4+, HLA-DR+, Ki67+, and langerin+ cellular subpopulations in colorectal and foreskin mucosa were similar in both groups. Thus, no greater trafficking of Ad5-specific CD4+ T lymphocytes to mucosal target sites was observed following rAd5 vaccination of rhesus monkeys with baseline Ad5 immunity. These findings from this nonhuman primate model provide evidence against the hypothesis that recruitment of vector-specific target cells to mucosal sites led to increased HIV-1 acquisition in Ad5-seropositive, uncircumcised vaccinees in the Step study. PMID:20686023

  2. Intranasal inoculation with an adenovirus vaccine encoding ten repeats of Aβ3-10 reduces AD-like pathology and cognitive impairment in Tg-APPswe/PSEN1dE9 mice.

    PubMed

    Li, Yu; Ma, Ying; Zong, Li-Xia; Xing, Xiao-Na; Guo, Rong; Jiang, Tong-Zi; Sha, Sha; Liu, Li; Cao, Yun-Peng

    2012-08-15

    To develop a safe and efficient vaccine for AD treatment, we constructed an adenovirus vector vaccine encoding ten repeats of Aβ3-10 and CpG motif as a molecular adjuvant. We demonstrated that therapeutic immunization with Ad-10×Aβ3-10-CpG elicits Aβ3-10 specific Th2-polarized immune response with high titers of anti-Aβ antibodies in APPswe/PSEN1dE9 mice, which in turn reduced Aβ deposits in brains and cognitive impairment. In addition, Ad-10×Aβ3-10-CpG reduced astrocytosis without increasing the incidence of microhemorrhage. Our findings of this study raise the possibility that the adenovirus vaccine Ad-10×Aβ3-10-CpG would be a safe and effective alternative for AD immunotherapy.

  3. Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer

    PubMed Central

    Wong, C M; Nash, L A; Del Papa, J; Poulin, K L; Falls, T; Bell, J C; Parks, R J

    2016-01-01

    When injected directly into a tumor mass, adenovirus (Ad) vectors only transduce cells immediately along the injection tract. Expression of fusogenic proteins from the Ad vector can lead to syncytium formation, which efficiently spreads the therapeutic effect. Fusogenic proteins can also cause cancer cell death directly, and enhance the release of exosome-like particles containing tumor-associated antigens, which boosts the anti-tumor immune response. In this study, we have examined whether delivery of an early region 1 (E1)-deleted, replication-defective Ad vector encoding the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can provide therapeutic efficacy in an immunocompetent mouse tumor model. A high multiplicity of infection of AdFAST is required to induce cell fusion in mouse mammary carcinoma 4T1 cells in vitro, and FAST protein expression caused a modest reduction in cell membrane integrity and metabolic activity compared with cells infected with a control vector. Cells expressing FAST protein released significantly higher quantities of exosomes. In immunocompetent Balb/C mice harboring subcutaneous 4T1 tumors, AdFAST did not induce detectable cancer cell fusion, promote tumor regression or prolong mouse survival compared with untreated mice. This study suggests that in the context of the 4T1 model, Ad-mediated FAST protein expression did not elicit a therapeutic effect. PMID:27740615

  4. Chimpanzee adenovirus and MVA-vectored respiratory syncytial virus vaccine is safe and expands humoral and cellular immunity in adults

    PubMed Central

    Green, CA; Scarselli, E; Sande, CJ; Thompson, AJ; de Lara, CM; Taylor, K; Haworth, K; Del Sorbo, M; Angus, B; Siani, L; Di Marco, S; Traboni, C; Folgori, A; Colloca, S; Capone, S; Vitelli, A; Cortese, R; Klenerman, P; Nicosia, A; Pollard, AJ

    2015-01-01

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication defective viral vectors encoding the RSV proteins F, N and M2-1 for the induction of humoral and cellular responses. We performed an open-label, dose-escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intra-muscular and intra-nasal administration of the adenoviral vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralising antibody titres rose in response to intramuscular (IM) prime with PanAd3-RSV, and after IM boost for individuals primed by the intra-nasal (IN) route. Circulating anti-F IgG and IgA antibody secreting cells (ASCs) were observed after IM prime and IM boost. RSV-specific T-cell responses were increased after IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. IFNγ secretion after boost was from both CD4+ and CD8+ T-cells, without detectable Th2 cytokines that have been previously associated with immune pathogenesis following exposure to RSV after formalin inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. PMID:26268313

  5. HSV-1 amplicon vectors elicit polyfunctional T cell responses to HIV-1 Env, and strongly boost responses to an adenovirus prime

    PubMed Central

    Duke, Cindy M.P.; Maguire, Casey A.; Keefer, Michael C.; Federoff, Howard J.; Bowers, William J.; Dewhurst, Stephen

    2007-01-01

    HSV-1 amplicon vectors elicit strong T-cell responses to encoded antigens but the qualitative nature of these responses is poorly understood. Antigen-specific CD4+ and CD8+ T-cell responses to amplicon and adenovirus (rAd5) vectors encoding HIV-1 gp120 were assessed following immunization of mice, by performing intracellular cytokine staining for IFNγ, IL-2 and TNFα, following stimulation of splenocytes with a HIV-1 Env peptide pool. The quality of the primary T-cell response to amplicon and rAd5 vectors was strikingly similar, but there were qualitative differences in responses to amplicon vectors that incorporated different promoters upstream of gp120 - suggesting that promoters can significantly influence immune response quality. When prime-boost combinations were studied, a rAd5 prime and amplicon boost elicited the highest T-cell response. Furthermore, protocols that incorporated a rAd5 prime consistently elicited a greater proportion of polyfunctional CD4+ T-cells - regardless of boost. This suggests that initial priming can shape immune response quality after a boost. Overall, these findings provide insight into effective vector combinations for HIV-1 vaccine development. PMID:17868958

  6. Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells.

    PubMed

    Xie, Hong; Hu, Jia; Pan, Huan; Lou, Yaxin; Lv, Ping; Chen, Yingyu

    2014-02-01

    FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non-small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment.

  7. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  8. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  9. Vaccine-Induced Immunity in Baboons by Using DNA and Replication-Incompetent Adenovirus Type 5 Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

    PubMed Central

    Casimiro, Danilo R.; Tang, Aimin; Chen, Ling; Fu, Tong-Ming; Evans, Robert K.; Davies, Mary-Ellen; Freed, Daniel C.; Hurni, William; Aste-Amezaga, Jose M.; Guan, Liming; Long, Romnie; Huang, Lingyi; Harris, Virginia; Nawrocki, Denise K.; Mach, Henryk; Troutman, Robert D.; Isopi, Lynne A.; Murthy, Krishna K.; Rice, Karen; Wilson, Keith A.; Volkin, David B.; Emini, Emilio A.; Shiver, John W.

    2003-01-01

    The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8+-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed. PMID:12805466

  10. Vector condensate and AdS soliton instability induced by a magnetic field

    NASA Astrophysics Data System (ADS)

    Cai, Rong-Gen; Li, Li; Li, Li-Fang; Wu, You

    2014-01-01

    We continue to study the holographic p-wave superconductor model in the Einstein-Maxwell-complex vector field theory with a non-minimal coupling between the complex vector field and the Maxwell field. In this paper we work in the AdS soliton background which describes a conformal field theory in the confined phase and focus on the probe approximation. We find that an applied magnetic field can lead to the condensate of the vector field and the AdS soliton instability. As a result, a vortex lattice structure forms in the spatial directions perpendicular to the applied magnetic field. As a comparison, we also discuss the vector condensate in the Einstein-SU(2) Yang-Mills theory and find that in the setup of the present paper, the Einstein-Maxwell-complex vector field model is a generalization of the SU(2) model in the sense that the vector field has a general mass and gyromagnetic ratio.

  11. An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen

    PubMed Central

    Jex, Edward; Feng, Tsungwei; Sivko, Gloria S.; Baillie, Leslie W.; Goldman, Stanley; Van Kampen, Kent R.; Tang, De-chu C.

    2013-01-01

    Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine. PMID:23100479

  12. CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter.

    PubMed

    Bhatia, Shilpa; O'Bryan, Samia M; Rivera, Angel A; Curiel, David T; Mathis, J Michael

    2016-01-01

    Ad vectors are promising delivery vehicles for cancer therapeutic interventions. However, their application is limited by promiscuous tissue tropism and hepatotoxicity. This limitation can be avoided by altering the native tropism of Ads so that they can be redirected to the target cells through alternate cellular receptors. The CXCR4 chemokine receptor belongs to a large superfamily of G-protein-coupled receptors and is known to be upregulated in a wide variety of cancers, including breast cancer and melanoma. These receptors have been associated with cancer cell survival, progression, and metastasis. In the current study, an Ad to cancer cells overexpressing CXCR4 by using a bispecific adapter, sCAR-CXCL12, was retargeted. The sCAR-CXCL12 adapter contained the soluble ectodomain form of the native Ad5 receptor (sCAR), which was fused to a mature human chemokine ligand, CXCL12, through a short peptide linker. A dramatic increase in the infectivity of cancer cells using a targeted Ad vector compared with an untargeted vector was observed. Furthermore, sCAR-CXCL12 attenuated Ad infection of liver ex vivo and in vivo and enhanced Ad vector infection of xenograft tumors implanted in immunodeficient SCID-bg mice. Thus, the sCAR-CXCL12 adapter could be used to retarget Ad vectors to chemokine receptor-positive tumors.

  13. CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter

    PubMed Central

    Bhatia, Shilpa; O’Bryan, Samia M; Rivera, Angel A; Curiel, David T; Mathis, J Michael

    2016-01-01

    Ad vectors are promising delivery vehicles for cancer therapeutic interventions. However, their application is limited by promiscuous tissue tropism and hepatotoxicity. This limitation can be avoided by altering the native tropism of Ads so that they can be redirected to the target cells through alternate cellular receptors. The CXCR4 chemokine receptor belongs to a large superfamily of G-protein-coupled receptors and is known to be upregulated in a wide variety of cancers, including breast cancer and melanoma. These receptors have been associated with cancer cell survival, progression, and metastasis. In the current study, an Ad to cancer cells overexpressing CXCR4 by using a bispecific adapter, sCAR-CXCL12, was retargeted. The sCAR-CXCL12 adapter contained the soluble ectodomain form of the native Ad5 receptor (sCAR), which was fused to a mature human chemokine ligand, CXCL12, through a short peptide linker. A dramatic increase in the infectivity of cancer cells using a targeted Ad vector compared with an untargeted vector was observed. Furthermore, sCAR-CXCL12 attenuated Ad infection of liver ex vivo and in vivo and enhanced Ad vector infection of xenograft tumors implanted in immunodeficient SCID-bg mice. Thus, the sCAR-CXCL12 adapter could be used to retarget Ad vectors to chemokine receptor-positive tumors. PMID:27957479

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

    PubMed Central

    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

  15. Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity.

    PubMed

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; Yi, Shaoqiong; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei

    2014-02-01

    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.

  16. Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

    PubMed Central

    Schwarzenberger, P; Hunt, J D; Robert, E; Theodossiou, C; Kolls, J K

    1997-01-01

    To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA. PMID:9343214

  17. Suppression effect of recombinant adenovirus vector containing hIL-24 on Hep-2 laryngeal carcinoma cells.

    PubMed

    Chen, Xuemei; Liu, DI; Wang, Junfu; Su, Qinghong; Zhou, Peng; Liu, Jinsheng; Luan, Meng; Xu, Xiaoqun

    2014-03-01

    The melanoma differentiation-associated gene-7 [MDA-7; renamed interleukin (IL)-24] was isolated from human melanoma cells induced to terminally differentiate by treatment with interferon and mezerein. MDA-7/IL-24 functions as a multimodality anticancer agent, possessing proapoptotic, antiangiogenic and immunostimulatory properties. All these attributes make MDA-7/IL-24 an ideal candidate for cancer gene therapy. In the present study, the human MDA-7/IL-24 gene was transfected into the human laryngeal cancer Hep-2 cell line and human umbilical vein endothelial cells (HUVECs) with a replication-incompetent adenovirus vector. Reverse transcription polymerase chain reaction and western blot analysis confirmed that the Ad-hIL-24 was expressed in the two cells. The expression of the antiapoptotic gene, Bcl-2, was significantly decreased and the IL-24 receptor was markedly expressed in Hep-2 cells following infection with Ad-hIL-24, but not in HUVECs. In addition, the expression of the proapoptotic gene, Bax, was induced and the expression of caspase-3 was increased in the Hep-2 cells and HUVECs. Methyl thiazolyl tetrazolium assay indicated that Ad-hIL-24 may induce growth suppression in Hep-2 cells but not in HUVECs. In conclusion, Ad-hIL-24 selectively inhibits proliferation and induces apoptosis in Hep-2 cells. No visible damage was found in HUVECs. Therefore, the results of the current study indicated that Ad-hIL-24 may have a potent suppressive effect on human laryngeal carcinoma cell lines, but is safe for healthy cells.

  18. Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets.

    PubMed

    Bilbao, Guadalupe; Contreras, Juan L; Dmitriev, Igor; Smyth, Cheryl A; Jenkins, Stacie; Eckhoff, Devin; Thomas, Francis; Thomas, Judith; Curiel, David T

    2002-03-01

    The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80

  19. Long-term correction of glycogen storage disease type II with a hybrid Ad-AAV vector.

    PubMed

    Sun, Bao-dong; Chen, Y-T; Bird, Andrew; Amalfitano, Andrea; Koeberl, Dwight D

    2003-02-01

    We administered an adenovirus-adeno-associated virus (Ad-AAV) vector encoding human acid alpha-glucosidase (hGAA) to acid alpha-glucosidase-knockout (GAA-KO) mice on day 3 of life by gastrocnemius injection. In contrast to previous results for muscle-targeted Ad vector in adult GAA-KO mice, the muscles of the hindlimb showed reduced glycogen content and persistent hGAA for as long as 6 months after neonatal Ad-AAV vector administration. Not only the injected gastrocnemius muscles, but also the hamstrings and quadriceps muscles produced therapeutic levels of hGAA as a result of widespread transduction with the Ad-AAV vector; moreover, hGAA activity was 50-fold elevated as compared to normal mice. Vector RNA was detected in the hindlimb muscles, the hearts, and the livers by northern blot analysis and/or by RT-PCR for as long as 6 months. The low levels of hGAA detected in the heart were attributable to transduction with the Ad-AAV vector, not to secretion of hGAA by the injected muscle and uptake by the heart. Finally, although an antibody response to hGAA was present, it did not prevent the correction of glycogen storage in the skeletal muscle of GAA-KO mice.

  20. Safety of local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions.

    PubMed

    Harvey, Ben-Gary; Maroni, Jaman; O'Donoghue, Kelley A; Chu, Karen W; Muscat, Jolene C; Pippo, Allison L; Wright, Connie E; Hollmann, Charleen; Wisnivesky, Juan P; Kessler, Paul D; Rasmussen, Henrik S; Rosengart, Todd K; Crystal, Ronald G

    2002-01-01

    To help define the safety profile of the use of adenovirus (Ad) gene transfer vectors in humans, this report summarizes our experience since April 1993 of the local administration of E1(-)/E3(-) Ad vectors to humans using low (<10(9) particle units) or intermediate (10(9)-10(11) particle units) doses. Included in the study are 90 individuals and 12 controls, with diverse comorbid conditions, including cystic fibrosis, colon cancer metastatic to liver, severe coronary artery disease, and peripheral vascular disease, as well as normals. These individuals received 140 different administrations of vector, with up to seven administrations to a single individual. The vectors used include three different transgenes (human cystic fibrosis transmembrane conductance regulator cDNA, E. coli cytosine deaminase gene, and the human vascular endothelial growth factor 121 cDNA) administered by six different routes (nasal epithelium, bronchial epithelium, percutaneous to solid tumor, intradermal, epicardial injection of the myocardium, and skeletal muscle). The total population was followed for 130.4 patient-years. The study assesses adverse events, common laboratory tests, and long-term follow-up, including incidence of death or development of malignancy. The total group incidence of major adverse events linked to an Ad vector was 0.7%. There were no deaths attributable to the Ad vectors per se, and the incidence of malignancy was within that expected for the population. Overall, the observations are consistent with the concept that local administration of low and intermediate doses of Ad vectors appears to be well tolerated.

  1. Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors.

    PubMed

    Sargent, K L; Ng, P; Evelegh, C; Graham, F L; Parks, R J

    2004-03-01

    Helper-dependent adenovirus vectors (hdAd), which are deleted of all viral protein-coding sequences, can mediate long-term expression of a therapeutic transgene and lead to life-long, phenotypic correction in animal models of genetic disease. Here, we describe a new system for the generation of hdAd, which utilizes the DNA size restrictions imposed on an Ad virion deleted of protein IX (pIX): such virions are reported to package up to only approximately 35 kb of viral DNA. A pIX(-) helper virus (approximately 37.3 kb) was easily grown on complementing 293pIX cells. Upon infection of noncomplementing cells, this virus was not capable of forming infectious virions, but provided replicative and packaging functions for propagation of a 30-kb hdAd. The pIX(-) helper virus was effective in amplifying an hdAd and, in combination with Cre-mediated excision in the viral-packaging signal, resulted in a 1000-fold reduction in helper virus contamination in hdAd stocks compared to Cre/lox alone, as determined by plaque assay. However, through slot blot analysis of DNA isolated from virions, we determined that the ratio of hdAd to helper DNA was 500:1, similar to the ratio observed when using Cre/lox alone. Surprisingly, a large amount of the 37.3-kb helper DNA was being packaged into the pIX-deleted virions, but these virions were incapable of establishing productive infections in plaque assays, for reasons which are still unclear. Nevertheless, the pIX(-) hdAd generated in this system infected cells and expressed a transgene at levels similar to those obtained with a pIX(+) hdAd. These data suggest that, although further studies are necessary to characterize the nature of the defective helper virions formed in this system, deletion of pIX from the helper virus genome does provide an effective method to prevent recovery of functional helper virus during hdAd amplification.

  2. Replicating adenovirus vector prime/protein boost strategies for HIV vaccine development

    PubMed Central

    Patterson, L. Jean; Robert-Guroff, Marjorie

    2008-01-01

    Background In the last few years the HIV vaccine field has moved forward a number of promising vaccine candidates into human clinical trials. Objective In this review we briefly discuss the advances made in vaccine development and HIV pathogenesis and give an overview of the body of work our lab has generated in multiple animal models on replication-competent Ad recombinant vaccines. Methods Emphasis is placed on comparative examination of vaccine components, routes of immunization and challenge models using replicating Ad vectors. Results/conclusion The overall findings make the case that replicating Ad vectors are superior in priming multiple arms of the immune system, and in conjunction with protein boosting, have resulted in dramatic protective efficacy leading to their advancement to phase 1 trials. Implications of the recent halting of the Merck Ad5-HIV phase 2b clinical trial for our vaccine approach and other vectored vaccines are discussed. PMID:18694354

  3. Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

    PubMed

    Gowen, Brian B; Ennis, Jane; Russell, Andrew; Sefing, Eric J; Wong, Min-Hui; Turner, Jeffrey

    2011-01-01

    Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

  4. A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

    PubMed

    Puntel, Mariana; Muhammad, A K M G; Candolfi, Marianela; Salem, Alireza; Yagiz, Kader; Farrokhi, Catherine; Kroeger, Kurt M; Xiong, Weidong; Curtin, James F; Liu, Chunyan; Bondale, Niyati S; Lerner, Jonathan; Pechnick, Robert N; Palmer, Donna; Ng, Philip; Lowenstein, Pedro R; Castro, Maria G

    2010-06-01

    Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

  5. Single dose of an adenovirus vectored mouse interferon-α protects mice from lethal EV71 challenge.

    PubMed

    Sun, Jialei; Ennis, Jane; Turner, Jeffrey D; Chu, Justin Jang Hann

    2016-10-01

    Enterovirus 71 (EV71) causes hand-foot-and-mouth diseases as well as neurological complications in young children. Interferon (IFN) can inhibit the replication of many viruses with low cytotoxic effects. Previously, an adenovirus vectored mouse interferon-α (DEF201), subtype 5, was generated by Wu et al, 2007. In this study, the antiviral effects of DEF201 against EV71 were evaluated in a murine model. 6-day-old BALB/c mice were administered a single dose of DEF201 before or after infection with lethal dose of EV71. The survival rate, clinical symptoms, tissue viral loads and histology pathogenesis were evaluated. IFN gene expression following a single dose of DEF201 maintained high concentrations of 100-9000 pg/mL for more than 7 days in mice serum. Pre-infection administration of a single dose of 10(6) PFU of DEF201 offered full protection of the mice against EV71 infection compared with the empty Ad5 vector control. In addition, virus load in DEF201-treated mice muscle tissue was significantly decreased as compared with empty vector control. Histopathology analysis revealed that DEF201 significantly prevented the development of severe tissue damage with reduction of viral antigen in the murine muscle tissue. Post-infection treatment at 6 h offered full protection and partial protection at 12 h, indicating that DEF201 could be used as an anti-EV71 therapeutic agent in early stage of EV71 infection. In addition, our study showed that DEF201 enhanced the neutralization ability of serum in EV71-vaccinated mice, implying that DEF201 could promote the production of specific anti-EV71 antibodies. In conclusion, single dose of DEF201 is highly efficacious as a prophylactic agent against EV71 infection in vivo.

  6. Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.

    PubMed

    Corjon, Stéphanie; Wortmann, Andreas; Engler, Tatjana; van Rooijen, Nico; Kochanek, Stefan; Kreppel, Florian

    2008-11-01

    Adenovirus (Ad) vector targeting requires presentation of specific ligands on the virion's surface. Geneti-chemical targeting is based on the genetic introduction of cysteine residues bearing reactive thiol groups into solvent-accessible capsomeres of the virion and subsequent chemical coupling of ligands. Here, we exploited this technology to modify the pIX capsomere with high-affinity ligands. Genetic introduction of C-terminal cysteines to pIX allowed for specific coupling of full-length proteins to the virion, while not affecting vector production. Direct comparison of the two high-affinity ligands receptor- associated protein (RAP) and transferrin (Tf) revealed that targeting after coupling of a high-affinity ligand to pIX presumably requires release of the ligand from its receptor after cell entry. In addition, data obtained by live cell imaging of labeled vector particles demonstrated that coupling of very large proteins to pIX can impair intracellular vector particle trafficking. Finally, we demonstrate that the geneti-chemical targeting technology is suitable for in vivo targeting to liver after intravenous injection. Our data provide significant insight into basic requirements for successful targeting of pIX-modified Ad vectors.

  7. Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions.

    PubMed

    Crystal, Ronald G; Harvey, Ben-Gary; Wisnivesky, Juan P; O'Donoghue, Kelley A; Chu, Karen W; Maroni, Jaman; Muscat, Jolene C; Pippo, Allison L; Wright, Connie E; Kaner, Robert J; Leopold, Philip L; Kessler, Paul D; Rasmussen, Henrik S; Rosengart, Todd K; Hollmann, Charleen

    2002-01-01

    In this study we analyze the adverse events and abnormal laboratory parameters following local administration of low (<10(9) particle units) and intermediate (10(9)-10(11) particle units) single and repetitive doses (140 total) of E1(-)E3(-) adenovirus (Ad) gene transfer vectors administered to the respiratory epithelium, solid tumors, skin, myocardium, and skeletal muscle in eight gene transfer trials since April 1993. In the accompanying paper by Harvey et al., (Hum. Gene Ther. 2002; 13:15-63), we conclude that for the total group, no deaths were attributable to the Ad vectors per se, and the incidence of major adverse events likely caused by an Ad vector was 0.7%. The present study analyzes the trials as a group to evaluate risk factors for the adverse events, abnormal values among laboratory parameters, and known deaths. Ten putative risk factors were assessed, including "patient-related" (age, sex, comorbid index and pretherapy anti-Ad antibodies), "vector-related" (dose, route, transgene, and number of vector administrations), and "trial-related" (trial in which the individual was enrolled, and whether surgery was part of the trial). While assessment of each factor individually suggested several possible associations with adverse events, abnormal laboratory parameters, or deaths, multivariate analysis identified only age, comorbid index, and surgery (comorbid index for death; age and surgery for non-death adverse events) as variables significantly associated with increased risk for a major (severity scale 3-4 of 4) adverse event for individuals enrolled in these gene transfer trials. Importantly, multivariate analysis suggested that vector-related parameters, including dose, route, transgene, or number of vector administrations at the doses and routes evaluated in these studies, do not appear to be significant risk factors for a major adverse event. With the caveat that these are phase I, uncontrolled trials, we conclude that (1) there is no definitive risk

  8. Preclinical safety assessment of Ad[I/PPT-E1A], a novel oncolytic adenovirus for prostate cancer.

    PubMed

    Schenk, Ellen; Essand, Magnus; Kraaij, Robert; Adamson, Rachel; Maitland, Norman J; Bangma, Chris H

    2014-03-01

    Prostate cancer is the most common malignancy in the Western world. Patients can be cured only when the tumor has not metastasized outside the prostate. However, treatment with curative intent fails in a significant number of men, often resulting in untreatable progressive disease with a fatal outcome. Oncolytic adenovirus therapy may be a promising adjuvant treatment to reduce local failure or the outgrowth of micrometastatic disease. Within the European gene therapy consortium GIANT, we have developed a novel prostate-specific oncolytic adenovirus: Ad[I/PPT-E1A]. This adenovirus specifically kills prostate cells via prostate-specific replication. This article describes the clinical development of Ad[I/PPT-E1A] with particular reference to the preclinical safety assessment of this novel virus. The preclinical safety assessment involved an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells, and a toxicity study in normal mice. These studies confirmed that Ad[I/PPT-E1A] efficiently kills prostate tumor cells in vivo, is not harmful to other organs, and is well tolerated in mice after systemic delivery. The safety, as well as the immunological effects of Ad[I/PPT-E1A] as a local adjuvant therapy, will now be studied in a phase I dose-escalating trial in patients with localized prostate cancer who are scheduled for curative radical prostatectomy and can be used as an updated paradigm for similar therapeutic viruses.

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

    PubMed

    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.

  10. Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine.

    PubMed

    Fernandez-Sainz, Ignacio; Medina, Gisselle N; Ramirez-Medina, Elizabeth; Koster, Marla J; Grubman, Marvin J; de Los Santos, Teresa

    2017-02-01

    A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 10(7) pfu/animal while a dose of 4×10(7)pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine. Published by Elsevier Inc.

  11. Tight positive regulation of transgene expression by a single adenovirus vector containing the rtTA and tTS expression cassettes in separate genome regions.

    PubMed

    Mizuguchi, Hiroyuki; Xu, Zhi-Li; Sakurai, Fuminori; Mayumi, Tadanori; Hayakawa, Takao

    2003-09-01

    We previously developed single adenovirus (Ad) vectors that contained the components for a tetracycline-regulatable gene-expression system in the E1 and E3 deletion regions, and showed that the Ad vectors containing the tet-on system exhibit a much inferior regulation of transgene expression than those containing the tet-off system. In many cases, the tet-on system may be preferable because of its positive regulation of transgene expression. To this end, in the present study, by introducing the latest generation reverse tetracycline-responsive transcriptional activator (rtTA2s-M2 or rtTA2s-S2) and the tetracycline-controlled transcriptional silencer (tTS) into the original tet-on system, we constructed various modified Ad-mediated tet-on systems. Among them, the novel single Ad vector, which contained three heterologous gene-expression cassettes of the gene of interest, rtTA2s-S2, and tTS in the E1 deletion region, the E3 deletion region, and the region between E4 and 3'ITR, respectively, displayed vastly improved doxycycline-inducible gene expression in terms of low basal expression, high induced expression, and high responsiveness to doxycycline both in vitro and in vivo. These results also suggest that the low responsiveness to doxycycline may explain why the original tet-on system in the context of the Ad vector is not effective in vivo. This is the first report describing the cloning of three heterologous gene-expression cassettes into three separate regions of the E1/E3-deleted Ad genome. This improved Ad-mediated tet-on system might be useful for gene therapy and greatly facilitate the analyses of gene function.

  12. Optimization and internalization mechanisms of PEGylated adenovirus vector with targeting peptide for cancer gene therapy.

    PubMed

    Yao, Xing-Lei; Yoshioka, Yasuo; Ruan, Gui-Xin; Chen, Yu-Zhe; Mizuguchi, Hiroyuki; Mukai, Yohei; Okada, Naoki; Gao, Jian-Qing; Nakagawa, Shinsaku

    2012-08-13

    We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

  13. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    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.

  14. Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

    PubMed Central

    Alexander, Jeff; Mendy, Jason; Vang, Lo; Avanzini, Jenny B.; Garduno, Fermin; Manayani, Darly J.; Ishioka, Glenn; Farness, Peggy; Ping, Li-Hua; Swanstrom, Ronald; Parks, Robert; Liao, Hua-Xin; Haynes, Barton F.; Montefiori, David C.; LaBranche, Celia; Smith, Jonathan; Gurwith, Marc; Mayall, Tim

    2013-01-01

    Background There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. Methods The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. Results Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. Conclusions The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical

  15. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

    Adenoviruses are associated with numerous disease outbreaks, particularly those involving d-cares, schools, children's camps, hospitals and other health care centers, and military settings. In addition, adenoviruses have been responsible for many recreational water outbreaks, including a great number of swimming pool outbreaks than any other waterborne virus (Gerba and Enriquez 1997). Two drinking water outbreaks have been documented for adenovirus (Divizia et al. 2004; Kukkula et al. 1997) but none for food. Of the 51 known adenovirus serotypes, one third are associated with human disease, while other infections are asymptomatic. Human disease associated with adenovirus infections include gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (Table 2). Children and the immunocompromised are more severely impacted by adenovirus infections. Subsequently, adenovirus is included in the EPA's Drinking Water Contaminant Candidate List (CCL), which is a list of unregulated contaminants found in public water systems that may pose a risk to public health (National Research Council 1999). Adenoviruses have been detected in various waters worldwide including wastewater, river water, oceans, and swimming pools (Hurst et al. 1988; Irving and Smith 1981; Pina et al. 1998). Adenoviruses typically outnumber the enteroviruses, when both are detected in surface waters. Chapron et al. (2000) found that 38% of 29 surface water samples were positive for infectious Ad40 and Ad41. Data are lacking regarding the occurrence of adenovirus in water in the US, particularly for groundwater and drinking water. Studies have shown, however, that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez et al. 1995), which may be due to their double-stranded DNA. Risk assessments have been conducted on waterborne adenovirus (Crabtree et al. 1997; van Heerden et al. 2005c). Using dose-response data for inhalation

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27628166

  17. Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

    PubMed Central

    Kim, Jin; Smith,*, Theodore; Idamakanti, Neeraja; Mulgrew, Kathy; Kaloss, Michele; Kylefjord, Helen; Ryan, Patricia C.; Kaleko, Michael; Stevenson, Susan C.

    2002-01-01

    Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo. PMID:11799184

  18. A complex adenovirus-vectored vaccine against Rift Valley fever virus protects mice against lethal infection in the presence of preexisting vector immunity.

    PubMed

    Holman, David H; Penn-Nicholson, Adam; Wang, Danher; Woraratanadharm, Jan; Harr, Mary-Katherine; Luo, Min; Maher, Ellen M; Holbrook, Michael R; Dong, John Y

    2009-11-01

    Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.

  19. Replication-incompetent adenovirus vector-mediated MDA-7/IL-24 selectively induces growth suppression and apoptosis of hepatoma cell Line SMMC-7721.

    PubMed

    Wang, Congjun; Xue, Xinbo; Yi, Jilin; Wu, Zaide; Chen, Kun; Zheng, Jianwei; Ji, Wenwei; Yu, Yuan

    2008-02-01

    In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture supernatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture supernatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the G2/M phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.

  20. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response.

    PubMed

    Moraes, Mauro Pires; Segundo, Fayna Diaz-San; Dias, Camila C; Pena, Lindomar; Grubman, Marvin J

    2011-11-28

    We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference

  1. Human adenovirus-vectored foot-and-mouth disease vaccines: establishment of a vaccine product profile through in vitro testing.

    PubMed

    Brake, D A; McIlhaney, M; Miller, T; Christianson, K; Keene, A; Lohnas, G; Purcell, C; Neilan, J; Schutta, C; Barrera, J; Burrage, T; Brough, D E; Butman, B T

    2012-01-01

    Next generation, foot-and-mouth disease (FMD) molecular vaccines based on replication deficient human adenovirus serotype 5 viral vectored delivery of FMD capsid genes (AdFMD) are being developed by the United States Dept. of Homeland Security and industry partners. The strategic goal of this program is to develop AdFMD licensed vaccines for the USA National Veterinary Stockpile for use, if needed, as emergency response tools during an FMD outbreak. This vaccine platform provides a unique opportunity to develop a set of in vitro analytical parameters to generate an AdFMD vaccine product profile to replace the current lot release test for traditional, inactivated FMD vaccines that requires FMDV challenge in livestock. The possibility of an indirect FMD vaccine potency test based on a serological alternative was initially investigated for a lead vaccine candidate, Adt.A24. Results show that serum virus neutralization (SVN) based serology testing for Adt.A24 vaccine lot release is not feasible, at least not in the context of vaccine potency assessment at one week post-vaccination. Thus, an in vitro infectious titer assay (tissue culture infectious dose 50, TCID50) which measures FMD infectious (protein expression) titer was established. Pre-validation results show acceptable assay variability and linearity and these data support further studies to validate the TCID50 assay as a potential potency release test. In addition, a quantitative physiochemical assay (HPLC) and three immunochemical assays (Fluorescent Focus-Forming Unit (FFU); tissue culture expression dose 50 (TCED50); Western blot) were developed for potential use as in vitro assays to monitor AdFMD vaccine lot-to-lot consistency and other potential applications. These results demonstrate the feasibility of using a traditional modified-live vaccine virus infectivity assay in combination with a set of physiochemical and immunochemical tests to build a vaccine product profile that will ensure the each Ad

  2. Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

    PubMed

    Gonçalves, Manuel A F V; Holkers, Maarten; van Nierop, Gijsbert P; Wieringa, Roeland; Pau, Maria G; de Vries, Antoine A F

    2008-08-29

    A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further

  3. Tumor Vascular Targeted Delivery of Polymer-conjugated Adenovirus Vector for Cancer Gene Therapy

    PubMed Central

    Yao, Xinglei; Yoshioka, Yasuo; Morishige, Tomohiro; Eto, Yusuke; Narimatsu, Shogo; Kawai, Yasuaki; Mizuguchi, Hiroyuki; Gao, Jian-Qing; Mukai, Yohei; Okada, Naoki; Nakagawa, Shinsaku

    2011-01-01

    Previously, we generated a cancer-specific gene therapy system using adenovirus vectors (Adv) conjugated to polyethylene glycol (Adv-PEG). Here, we developed a novel Adv that targets both tumor tissues and tumor vasculatures after systemic administration by conjugating CGKRK tumor vasculature homing peptide to the end of a 20-kDa PEG chain (Adv-PEGCGKRK). In a primary tumor model, systemic administration of Adv-PEGCGKRK resulted in ~500- and 100-fold higher transgene expression in tumor than that of unmodified Adv and Adv-PEG, respectively. In contrast, the transgene expression of Adv-PEGCGKRK in liver was about 400-fold lower than that of unmodified Adv, and was almost the same as that of Adv-PEG. We also demonstrated that transgene expression with Adv-PEGCGKRK was enhanced in tumor vessels. Systemic administration of Adv-PEGCGKRK expressing the herpes simplex virus thymidine kinase (HSVtk) gene (Adv-PEGCGKRK-HSVtk) showed superior antitumor effects against primary tumors and metastases with negligible side effects by both direct cytotoxic effects and inhibition of tumor angiogenesis. These results indicate that Adv-PEGCGKRK has potential as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both primary tumors and metastases. PMID:21673661

  4. Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX ▿

    PubMed Central

    Poulin, Kathy L.; Lanthier, Robert M.; Smith, Adam C.; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L.; O'Meara, Ryan W.; Kothary, Rashmi; Lorimer, Ian A.; Parks, Robin J.

    2010-01-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions. PMID:20631131

  5. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    PubMed

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

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

    PubMed

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    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.

  7. Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

    PubMed Central

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    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. PMID:24124483

  8. Pre-clinical safety assessment of Ad[I/PPT-E1A], a novel oncolytic adenovirus for prostate cancer.

    PubMed

    Schenk, Ellen; Essand, Magnus; Kraaij, Robert; Adamson, Rachel; Maitland, Norman; Bangma, Chris H

    2014-02-18

    Prostate cancer is the most common malignancy in the Western world. Patients can only be cured when the tumour has not metastasised outside the prostate. However, treatment with curative intent fails in a significant number of men, often resulting in untreatable progressive disease with a fatal outcome. Oncolytic adenovirus therapy may be a promising adjuvant treatment to reduce local failure or the outgrowth of micrometastatic disease. Within the European gene therapy consortium GIANT, we have developed a novel prostate-specific oncolytic adenovirus: Ad[I/PPT-E1A] that specifically kills prostate cells via prostate-specific replication. This paper describes the clinical development of Ad[I/PPT-E1A] with particular reference to the pre-clinical safety assessment of this novel virus. The pre-clinical safety assessment involved an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells and a toxicity study in normal mice. These studies confirmed that Ad[I/PPT-E1A] efficiently kills prostate tumour cells in vivo, is not harmful to other organs and is well-tolerated in mice after systemic delivery. The safety, as well as the immunological effects of Ad[I/PPT-E1A] as a local adjuvant therapy will now be studied in a phase I dose-escalating trial in patients with localised prostate cancer who are scheduled for curative radical prostatectomy and can be used as an updated paradigm for similar therapeutic viruses.

  9. Canine adenovirus based rabies vaccines.

    PubMed

    Tordo, N; Foumier, A; Jallet, C; Szelechowski, M; Klonjkowski, B; Eloit, M

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control.

  10. Development of replication-deficient adenovirus malaria vaccines.

    PubMed

    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.

  11. Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration.

    PubMed

    Fernández, Erlinda; Toledo, Jorge R; Chiong, Maylin; Parra, Francisco; Rodríguez, Elsa; Montero, Carlos; Méndez, Lídice; Capucci, Lorenzo; Farnós, Omar

    2011-08-15

    Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.

  12. Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083

    PubMed Central

    Walsh, Stephen R.; Moodie, Zoe; Fiore-Gartland, Andrew J.; Morgan, Cecilia; Wilck, Marissa B.; Hammer, Scott M.; Buchbinder, Susan P.; Kalams, Spyros A.; Goepfert, Paul A.; Mulligan, Mark J.; Keefer, Michael C.; Baden, Lindsey R.; Swann, Edith M.; Grant, Shannon; Ahmed, Hasan; Li, Fusheng; Hertz, Tomer; Self, Steven G.; Friedrich, David; Frahm, Nicole; Liao, Hua-Xin; Montefiori, David C.; Tomaras, Georgia D.; McElrath, M. Juliana; Hural, John; Graham, Barney S.; Jin, Xia

    2016-01-01

    Background. Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. Methods. A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. Results. T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6–1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2–5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). Conclusions. These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. Clinical Trials Registration. NCT01095224. PMID:26475930

  13. Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor.

    PubMed

    Brokhman, Irina; Pomp, Oz; Fishman, Lital; Tennenbaum, Tamar; Amit, Michal; Itzkovitz-Eldor, Joseph; Goldstein, Ronald S

    2009-04-01

    Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.

  14. Adenovirus vector E4 gene regulates connexin 40 and 43 expression in endothelial cells via PKA and PI3K signal pathways.

    PubMed

    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

    2005-05-13

    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 intra-tracheal 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.

  15. Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector.

    PubMed

    Serratrice, Nicolas; Cubizolle, Aurelie; Ibanes, Sandy; Mestre-Francés, Nadine; Bayo-Puxan, Neus; Creyssels, Sophie; Gennetier, Aurelie; Bernex, Florence; Verdier, Jean-Michel; Haskins, Mark E; Couderc, Guilhem; Malecaze, Francois; Kalatzis, Vasiliki; Kremer, Eric J

    2014-05-10

    Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.

  16. Safety studies on an adenovirus recombinant vaccine for rabies (AdRG1.3-ONRAB) in target and non-target species.

    PubMed

    Knowles, M Kimberly; Nadin-Davis, Susan A; Sheen, Mary; Rosatte, Rick; Mueller, Rudi; Beresford, Andrew

    2009-11-05

    A replication-competent human adenovirus vector in which the rabies virus glycoprotein gene was inserted (AdRG1.3-ONRAB) was given by direct instillation into the oral cavity to representatives of three wildlife vector species of concern in Ontario (red fox, raccoon and striped skunk) and to a variety of non-target wildlife species, domestic and laboratory species. Despite use of a relatively high dose of vaccine, no untoward clinical signs were observed. Subsequent to vaccine exposure, detection of vaccine virus in lung, spleen, intestine, liver, kidney and brain of each animal was attempted using an ONRAB-specific assay combining PCR with Southern blotting (PCR-SB). Of the 1280 tissue samples obtained from vaccinates or contact animals, 18 (1.4%) were found to be PCR-SB positive. Virus isolation attempts were performed utilizing cell culture for all PCR-SB positive tissues and a selection of PCR-SB negative tissues. Histological examination performed on all PCR-SB positive tissues failed to identify lesions attributed to the vaccine. A quantitative real-time PCR was used to determine the excretion of the vaccine in feces and in the oral cavity with 0.8% of oral swabs and 6.8% of fecal specimens found to be positive. The low rates of recovery of vaccine virus from tissues, feces and the oral cavity suggest that the likelihood of ONRAB causing a negative impact on wildlife species is unlikely.

  17. Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-α-glucosidase

    PubMed Central

    Amalfitano, A.; McVie-Wylie, A. J.; Hu, H.; Dawson, T. L.; Raben, N.; Plotz, P.; Chen, Y. T.

    1999-01-01

    This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid α-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies. PMID:10430861

  18. Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

    PubMed Central

    Singh, Shailbala; Toro, Haroldo; Tang, De-Chu; Briles, Worthie E.; Yates, Linda M.; Kopulos, Renee T.; Collisson, Ellen W.

    2010-01-01

    Avian influenza virus (AIV) specific CD8+ T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8+ T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologousH7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8+ T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses. PMID:20557918

  19. Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins.

    PubMed

    Wu, Jie; Chen, Ke-DA; Gao, Meng; Chen, Gang; Jin, Su-Feng; Zhuang, Fang-Cheng; Wu, Xiao-Hong; Jiang, Yun-Shui; Li, Jian-Bo

    2015-04-01

    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×10(9) IU/ml and 3.0×10(9) IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 10(9) IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ(2)MSB=20.00 and χ(2)WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development.

  20. Intranasal Mucosal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis

    PubMed Central

    Xing, Zhou; McFarland, Christine T.; Sallenave, Jean-Michel; Izzo, Angelo; Wang, Jun; McMurray, David N.

    2009-01-01

    Background Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models. Methods and Findings Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge. Conclusions Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials. PMID:19516906

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

    PubMed Central

    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

  2. Spontaneous mutants of the adenovirus-simian virus 40 hybrid, Ad2/sup +/ND3, that grow efficiently in monkey cells

    SciTech Connect

    Anderson, C.W.

    1981-05-01

    An attempt was made to isolate spontaneous mutants of adenovirus type 2 and of the adenovirus-SV40 hybrids, Ad2/sup +/ND3 and Ad2/sup +/ND5, that would grow efficiently on monkey cells. Virus stocks were serially passaged through the semipermissive established monkey line CV-1. After five serial passages in the absence of intentional mutagenesis, only stocks of Ad2/sup +/ND3 yielded significant numbers of variants that plaqued with similar efficiency on human and on monkey cell monolayers. Four independent Ad2/sup +/ND3 variants, designated hr600, hr601, hr602, and hr603, have been isolated and partially characterized. No difference was found between the genomes of these variants and the genome of parental Ad2/sup +/ND3 by restriction enzyme analysis or by the analysis of heteroduplexes between Ad2/sup +/ND3 (or variant) DNA and DNA of the hybrid Ad2/sup +/ND1.

  3. Intranasal Adenovirus-Vectored Vaccine for Induction of Long-Lasting Humoral Immunity-Mediated Broad Protection against Influenza in Mice

    PubMed Central

    Kim, Eun Hye; Park, Hae-Jung; Han, Gye-Yeong; Song, Man-Ki; Pereboev, Alexander; Hong, Jeong S.; Chang, Jun; Byun, Young-Ho; Seong, Baik Lin

    2014-01-01

    ABSTRACT Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes. IMPORTANCE Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of

  4. Overexpression of the Adenovirus Type 12 (Ad12) pTP or E1A Gene Facilitates Ad12 DNA Replication in Nonpermissive BHK21 Hamster Cells

    PubMed Central

    Hösel, Marianna; Webb, Dennis; Schröer, Jörg; Schmitz, Birgit; Doerfler, Walter

    2001-01-01

    In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP. PMID:11581373

  5. Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production.

    PubMed

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

    2007-05-01

    Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established.

  6. Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35

    PubMed Central

    Zhang, Wen-feng; Shao, Hong-wei; Wu, Feng-lin; Xie, Xin; Li, Zhu-Ming; Bo, Hua-Ben; Shen, Han; Wang, Teng; Huang, Shu-lin

    2016-01-01

    Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy. PMID:26972139

  7. Biobased monoliths for adenovirus purification.

    PubMed

    Fernandes, Cláudia S M; Gonçalves, Bianca; Sousa, Margarida; Martins, Duarte L; Barroso, Telma; Pina, Ana Sofia; Peixoto, Cristina; Aguiar-Ricardo, Ana; Roque, A Cecília A

    2015-04-01

    Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

  8. A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

    PubMed

    Rubinchik, S; Wang, D; Yu, H; Fan, F; Luo, M; Norris, J S; Dong, J Y

    2001-11-01

    Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.

  9. A Phase I Double Blind, Placebo-Controlled, Randomized Study of a Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults

    PubMed Central

    Hayes, Peter; Gill, Dilbinder; Kopycinski, Jakub; Cheeseman, Hannah; Cashin-Cox, Michelle; Naarding, Marloes; Clark, Lorna; Fernandez, Natalia; Bunce, Catherine A.; Hay, Christine M.; Welsh, Sabrina; Komaroff, Wendy; Hachaambwa, Lottie; Tarragona-Fiol, Tony; Sayeed, Eddy; Zachariah, Devika; Ackland, James; Loughran, Kelley; Barin, Burc; Cormier, Emmanuel; Cox, Josephine H.; Fast, Patricia; Excler, Jean-Louis

    2012-01-01

    Background 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. Methods 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 2×109 (A), 2×1010 (B), 2×1011 (C), or Ad35-GRIN 1×1010 (D) viral particles. Results 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 106 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. Conclusion/Significance 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

  10. [Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells].

    PubMed

    Yin, Kai; Ma, Li; Shen, Chuan'an; Shang, Yuru; Li, Dawei; Li, Longzhu; Zhao, Dongxu; Cheng, Wenfeng

    2016-05-01

    To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture

  11. Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses III. Base Composition, Molecular Weight, and Conformation of the Ad2+ND1 Genome

    PubMed Central

    Crumpacker, Clyde S.; Henry, Patrick H.; Kakefuda, Tuyoski; Rowe, Wallace P.; Levin, Myron J.; Lewis, Andrew M.

    1971-01-01

    The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2+ND1, differs from the defective Ad-SV40 hybrid populations previously described, in that this hybrid virus can replicate without the aid of nonhybrid adenovirus helper. Consequently, the deoxyribonucleic acid (DNA) from this virus, which can be obtained free of nonhybrid adenovirus DNA, is well suited for biophysical studies on Ad-SV40 hybrid DNA. Such studies have been performed and demonstrate Ad2+ND1 DNA to have a buoyant density (1.715 g/cm3) and thermal denaturation profile (Tm = 75.1 C) almost identical with nonhybrid Ad2 DNA. Furthermore, its molecular weight, as determined by analytical zone sedimentation and electron microscopy, was 22 × 106 to 25 × 106 daltons, which is also very similar to that determined for Ad2. Electron micrographs showed all of the hybrid molecules to be double-stranded and linear. By using this determination of the molecular weight of Ad2+ND1 DNA and assuming that 1% of this molecule consists of covalently linked SV40 DNA (see companion paper), we calculate that the hybrid DNA molecule contains 220 × 103 to 250 × 103 daltons of SV40 DNA, or the equivalent of one-tenth of the SV40 genome. PMID:4323710

  12. Mucosal vaccination by adenoviruses displaying reovirus sigma 1.

    PubMed

    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

    2015-08-01

    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.

  13. Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using direct homologous challenge

    USDA-ARS?s Scientific Manuscript database

    The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularl...

  14. Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

    PubMed

    Bennett, David; Sakurai, Fuminori; Shimizu, Kahori; Matsui, Hayato; Tomita, Kyoko; Suzuki, Takayuki; Katayama, Kazufumi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2012-12-03

    In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

  15. Functionally inactivated dominant viral antigens of human cytomegalovirus delivered in replication incompetent adenovirus type 6 vectors as vaccine candidates.

    PubMed

    Tang, Aimin; Freed, Daniel C; Li, Fengsheng; Meschino, Steve; Prokop, Michael; Bett, Andrew; Casimiro, Danilo; Wang, Dai; Fu, Tong-Ming

    2017-05-11

    T cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

  16. TheQ1 Influence of Innate and Pre-Existing Immunity on Adenovirus Therapy

    PubMed Central

    Zaiss, Anne K.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2010-01-01

    Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed; Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre-existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad-immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre-existing immunity. PMID:19711370

  17. Evaluation of apoptogenic adenovirus type 5 oncolytic vectors in a Syrian hamster head and neck cancer model

    PubMed Central

    Subramanian, T.; Strebeck, Frank F.; West, Cheri L.; Varvares, Mark; Chinnadurai, G.

    2015-01-01

    Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9, 10-dimethyl-1, 2-benzanthracene (DMBA). These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55K (which are defective in the expression of either E1B-19K alone or both E1B-19K and E1B-55K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intra-tumoral administration of HAdV5 E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers. PMID:24874842

  18. Avian influenza in ovo vaccination with replication defective recombinant adenovirus in chickens: Vaccine potency, antibody persistence, and maternal antibody transfer

    USDA-ARS?s Scientific Manuscript database

    Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibo...

  19. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  20. Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene

    PubMed Central

    Putranto, Endy Widya; Kinoshita, Rie; Watanabe, Masami; Sadahira, Takuya; Murata, Hitoshi; Yamamoto, Ken-Ichi; Futami, Junichiro; Kataoka, Ken; Inoue, Yusuke; Winarsa Ruma, I. Made; Sumardika, I. Wayan; Youyi, Chen; Kubo, Miyoko; Sakaguchi, Yoshihiko; Saito, Kenji; Nasu, Yasutomo; Kumon, Hiromi; Huh, Nam-Ho; Sakaguchi, Masakiyo

    2017-01-01

    Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5′ located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5′ located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy. PMID:28693271

  1. Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene.

    PubMed

    Putranto, Endy Widya; Kinoshita, Rie; Watanabe, Masami; Sadahira, Takuya; Murata, Hitoshi; Yamamoto, Ken-Ichi; Futami, Junichiro; Kataoka, Ken; Inoue, Yusuke; Winarsa Ruma, I Made; Sumardika, I Wayan; Youyi, Chen; Kubo, Miyoko; Sakaguchi, Yoshihiko; Saito, Kenji; Nasu, Yasutomo; Kumon, Hiromi; Huh, Nam-Ho; Sakaguchi, Masakiyo

    2017-07-01

    Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

  2. Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissues

    USDA-ARS?s Scientific Manuscript database

    Recombinant replication-defective human adenovirus type 5 (Ad5) vaccines containing capsid-coding regions from foot-and-mouth disease virus (FMDV) have been demonstrated to induce effective immune responses and provide homologous protective immunity against FMDV in cattle. However, basic mechanisms ...

  3. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

    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.

    2015-08-15

    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.

  4. Anti-tumor efficacy of a transcriptional replication-competent adenovirus, Ad-OC-E1a, for osteosarcoma pulmonary metastasis.

    PubMed

    Li, Xiong; Jung, Chaeyong; Liu, You-Hong; Bae, Kyung-Hee; Zhang, Yan-Ping; Zhang, Hong-Ji; Vanderputten, Dale; Jeng, Meei-Huey; Gardner, Thomas A; Kao, Chinghai

    2006-06-01

    Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor-specific manner are a potential modality for OSPM therapy. We detected OC mRNA expression by RNA in situ hybridization in OSA and OSPM samples from patients, and tested OC promoter transcriptional activity in OSA and non-OSA cell lines. Then we used a transcriptional replication-competent adenovirus, Ad-OC-E1a, to treat OSPM, and evaluated its tumor-specific replication and killing activities in vitro as well as anti-OSPM efficacy in vivo via systemic delivery. OC mRNA was detected in all types of OSA tissues, including OSPM tissues. The transcriptional activity of the OC promoter was much higher in a OSPM cell line SAOS-2LM7 and primary OSA cell line MG63 than in non-OSA cell lines, including cell lines from breast cancer, colon cancer, and liver cancer. Ad-OC-E1a expressed E1a protein only in MG63 and SAOS-2LM7, which indicated that adenovirus E1a was under strict control by the OC promoter. Ad-OC-E1a demonstrated killing and viral replication activity close to wild-type adenovirus levels in MG63 and SAOS-2LM7, but the killing and viral replication activities were attenuated significantly in cells expressing low OC transcriptional activity. To test whether Ad-OC-E1a could be used to target human OSPM in vivo, SAOS-2LM7 pulmonary metastasis models in nude mice were induced and treated by tail-vein injection with Ad-OC-E1a. Compared to tumor nodules in the lung in groups treated with PBS or control virus, the quantity of metastasized tumor nodules decreased

  5. New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo.

    PubMed

    Rubinchik, S; Woraratanadharm, J; Yu, H; Dong, J-Y

    2005-03-01

    Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.

  6. Adding and Subtracting Vectors: The Problem with the Arrow Representation

    ERIC Educational Resources Information Center

    Heckler, Andrew F.; Scaife, Thomas M.

    2015-01-01

    A small number of studies have investigated student understanding of vector addition and subtraction in generic or introductory physics contexts, but in almost all cases the questions posed were in the vector arrow representation. In a series of experiments involving over 1000 students and several semesters, we investigated student understanding…

  7. Adding and Subtracting Vectors: The Problem with the Arrow Representation

    ERIC Educational Resources Information Center

    Heckler, Andrew F.; Scaife, Thomas M.

    2015-01-01

    A small number of studies have investigated student understanding of vector addition and subtraction in generic or introductory physics contexts, but in almost all cases the questions posed were in the vector arrow representation. In a series of experiments involving over 1000 students and several semesters, we investigated student understanding…

  8. Efficacy of helper-dependent adenovirus vector-mediated gene therapy in murine glycogen storage disease type Ia.

    PubMed

    Koeberl, Dwight D; Sun, B; Bird, A; Chen, Y T; Oka, K; Chan, L

    2007-07-01

    Genetic deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, also known as von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia and growth retardation. We tested whether helper-dependent adenovirus (HDAd)-mediated hepatic delivery of G6Pase would lead to prolonged survival and sustained correction of the metabolic abnormalities in G6Pase knockout (KO) mice, a model for a severe form of GSD-Ia. An HDAd vector encoding G6Pase was administered intravenously (2 or 5 x 10(12)vector particles/kg) to 2-week-old (w.o.) G6Pase-KO mice. Following HDAd vector administration survival was prolonged to a median of 7 months, in contrast to untreated affected mice that did not survive past 3 weeks of age. G6Pase levels increased more than tenfold between 3 days and 28 weeks after HDAd injection (P < 0.03). The weights of untreated 2 w.o. G6Pase-KO mice were approximately half those of their unaffected littermates, and treatment stimulated their growth to the size of wild-type mice. Severe hypoglycemia and hypercholesterolemia, which are hallmarks of GSD-Ia both in humans and in mice, were also restored to normalcy by the treatment. Glycogen accumulation in the liver was markedly reduced. The efficacy of HDAd-G6Pase treatment in reversing the physiological and biochemical abnormalities associated with GSD-Ia in affected G6Pase-KO mice justifies further preclinical evaluation in murine and canine models of GSD-Ia.

  9. Evaluation of Novel Large Cut-Off Ultrafiltration Membranes for Adenovirus Serotype 5 (Ad5) Concentration

    PubMed Central

    Peixoto, Cristina; Roederstein, Susanne; Schleuss, Tobias; Alves, Paula M.; Mota, José P. B.; Carrondo, Manuel J. T.

    2014-01-01

    The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed. PMID:25546428

  10. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses.

    PubMed

    Takagi-Kimura, M; Yamano, T; Tagawa, M; Kubo, S

    2014-03-01

    Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

  11. Efficacy and safety of a live canine adenovirus-vectored rabies virus vaccine in swine.

    PubMed

    Liu, Ye; Zhang, Shoufeng; Ma, Guangpeng; Zhang, Fei; Hu, Rongliang

    2008-10-03

    Rabies infections in swine have been reported occasionally in recent years in certain geographic locations. Although a protective vaccine consisting of inactivated rabies virus is available for use in swine, searching for a more economically viable formulation for use in developing countries is always a priority. This work describes the testing of a canine adenovirus that expresses a rabies viral epitope (CAV-2-E3Delta-RGP) in a porcine rabies model. The data presented here show that the recombinant viral vaccine was effective in protecting swine against rabies if administered intramuscularly, but not orally or intranasally, and that protection was probably related to the development of a humoral response that lasted at least 28 weeks. Following vaccination, no behavioral abnormalities were observed in vaccinated swine and virus particles were not detected in either tissues or body fluids, indicating that this formulation was safe. The recombinant virus stimulated an effective level of antibody response in the immunized swine after a single intramuscular inoculation.

  12. Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens.

    PubMed

    Sánchez Ramos, Oliberto; González Pose, Alain; Gómez-Puerta, Silvia; Noda Gomez, Julia; Vega Redondo, Armando; Águila Benites, Julio César; Suárez Amarán, Lester; Parra, Natalie C; Toledo Alonso, Jorge R

    2011-05-01

    Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.

  13. Comparison of efficiency between FLPe and Cre for recombinase-mediated cassette exchange in vitro and in adenovirus vector production.

    PubMed

    Takata, Yuki; Kondo, Saki; Goda, Naoki; Kanegae, Yumi; Saito, Izumu

    2011-07-01

    Cre and FLP recombinases mediate not only specific deletions and insertions, but also the recombinase-mediated cassette exchange (RMCE) reaction, which is used in cell biotechnology including ES cells and mouse genetics. However, comparison of efficiencies for Cre and FLP in RMCE has not been made. We here examined the detailed process of RMCE with Cre and FLP in vitro using mutant loxP 2272 and three mutant FRTs (FRT G, FRT H, and FRT F3) and then quantitatively compared the RMCE reactions in vitro. Interestingly, in the in vitro reactions, the RMCE efficiency of Cre reached a plateau level of approximately 5% and did not proceed further, whereas that of FLPe reached approximately 12-13%, showing that FLPe reached a higher level of efficiency than Cre possibly when they were supplied at a very high concentration. Moreover, we quantitatively compared the production efficiency of E1-deleted adenovirus vector using the RMCE method with Cre or FLP. The results showed that FLPe was again found more efficient than Cre in RMCE reaction. Thus, although Cre is considered more active than, or similar to, FLPe, it may not be necessarily true for RMCE reaction. Possible reasons explaining these results are discussed.

  14. Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adenovirus vector containing the CD4 gene.

    PubMed Central

    Yasukawa, M; Inoue, Y; Ohminami, H; Sada, E; Miyake, K; Tohyama, T; Shimada, T; Fujita, S

    1997-01-01

    It has been reported recently that CD4 is a major component of the receptor for human herpesvirus 7 (HHV-7), which has been newly identified as a T-lymphotropic virus. To investigate further the role of CD4 in HHV-7 infection, we examined the susceptibility to HHV-7 infection of various CD4-negative or weakly positive cell lines into which the cDNA for CD4 was transferred using an adenovirus vector (Adex1CACD4). Of 13 cell lines transduced with Adex1CACD4, including T-lymphoid, B-lymphoid, monocytoid, and myeloid cell lines, one T-lymphoid cell line, one monocytoid cell line, and two cell lines established from the blast crisis of chronic myelogenous leukemia showed high susceptibility to HHV-7 infection. Taken together with the results of previous studies, these data suggest strongly that CD4 is a major component of the binding receptor for HHV-7. This study also shows that HHV-7 may be able to infect CD4-positive hematopoietic precursor cells as well as T lymphocytes. PMID:8995705

  15. Six-Month Assessment of a Phase I Trial of Angiogenic Gene Therapy for the Treatment of Coronary Artery Disease Using Direct Intramyocardial Administration of an Adenovirus Vector Expressing the VEGF121 cDNA

    PubMed Central

    Rosengart, Todd K.; Lee, Leonard Y.; Patel, Shailen R.; Kligfield, Paul D.; Okin, Peter M.; Hackett, Neil R.; Isom, O. Wayne; Crystal, Ronald G.

    1999-01-01

    Objective To summarize the 6-month follow-up of a cohort of patients with clinically significant coronary artery disease who received direct myocardial injection of an E1−E3− adenovirus (Ad) gene transfer vector (AdGVVEGF121.10) expressing the human vascular endothelial growth factor (VEGF) 121 cDNA to induce therapeutic angiogenesis. Background Therapeutic angiogenesis describes a novel approach to the treatment of vascular occlusive disease that uses the administration of growth factors known to induce neovascularization of ischemic tissues. Methods Direct myocardial injection of AdGVVEGF121.10 into an area of reversible ischemia was carried out in 21 patients as an adjunct to conventional coronary artery bypass grafting (group A, n = 15) or as sole therapy using a minithoracotomy (group B, n = 6). Results No evidence of systemic or cardiac-related adverse events related to vector administration was observed up to 6 months after therapy. Trends toward improvement in angina class and exercise treadmill testing at 6-month follow-up in the sole therapy group suggest the effects of this therapy are persistent for ≥6 months. Conclusions This study suggests that direct myocardial administration of AdGVVEGF121.10 appears to be well tolerated in patients with clinically significant coronary artery disease. Initiation of phase II evaluation of this therapy appears warranted. PMID:10522716

  16. Capsid-like Arrays in Crystals of Chimpanzee Adenovirus Hexon

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

    The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 Angstroms, b = 433.0 Angstroms, c = 159.3 Angstroms, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 Angstroms resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.

  17. Applying Genomic and Bioinformatic Resources to Human Adenovirus Genomes for Use in Vaccine Development and for Applications in Vector Development for Gene Delivery

    PubMed Central

    Seto, Jason; Walsh, Michael P.; Mahadevan, Padmanabhan; Zhang, Qiwei; Seto, Donald

    2010-01-01

    Technological advances and increasingly cost-effect methodologies in DNA sequencing and computational analysis are providing genome and proteome data for human adenovirus research. Applying these tools, data and derived knowledge to the development of vaccines against these pathogens will provide effective prophylactics. The same data and approaches can be applied to vector development for gene delivery in gene therapy and vaccine delivery protocols. Examination of several field strain genomes and their analyses provide examples of data that are available using these approaches. An example of the development of HAdV-B3 both as a vaccine and also as a vector is presented. PMID:21994597

  18. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs

    PubMed Central

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude. PMID:23127366

  19. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

    PubMed

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

  20. Hexon Modification to Improve the Activity of Oncolytic Adenovirus Vectors against Neoplastic and Stromal Cells in Pancreatic Cancer

    PubMed Central

    Lucas, Tanja; Benihoud, Karim; Vigant, Frédéric; Schmidt, Christoph Q. Andreas; Simmet, Thomas; Kochanek, Stefan

    2015-01-01

    Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety. PMID:25692292

  1. Hexon modification to improve the activity of oncolytic adenovirus vectors against neoplastic and stromal cells in pancreatic cancer.

    PubMed

    Lucas, Tanja; Benihoud, Karim; Vigant, Frédéric; Schmidt, Christoph Q; Schmidt, Christoph Q Andreas; Wortmann, Andreas; Bachem, Max G; Simmet, Thomas; Kochanek, Stefan

    2015-01-01

    Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.

  2. Adding and subtracting vectors: The problem with the arrow representation

    NASA Astrophysics Data System (ADS)

    Heckler, Andrew F.; Scaife, Thomas M.

    2015-06-01

    A small number of studies have investigated student understanding of vector addition and subtraction in generic or introductory physics contexts, but in almost all cases the questions posed were in the vector arrow representation. In a series of experiments involving over 1000 students and several semesters, we investigated student understanding of vector addition and subtraction in both the arrow and algebraic notation (using i ^, j ^, k ^) in generic mathematical and physics contexts. First, we replicated a number of previous findings of student difficulties in the arrow format and discovered several additional difficulties, including the finding that different relative arrow orientations can prompt different solution paths and different kinds of mistakes, which suggests that students need to practice with a variety of relative orientations. Most importantly, we found that average performance in the i j k format was typically excellent and often much better than performance in the arrow format in either the generic or physics contexts. Further, while we find that the arrow format tends to prompt students to a more physically intuitive solution path, we also find that, when prompted, student solutions in the i j k format also display significant physical insights into the problem. We also find a hierarchy in correct answering between the two formats, with correct answering in the i j k format being more fundamental than for the arrow format. Overall, the results suggest that many student difficulties with these simple vector problems lie with the arrow representation itself. For instruction, these results imply that introducing the i j k notation (or some equivalent) with the arrow notation concurrently may be a very useful way to improve student performance as well as help students to learn physics concepts involving vector addition and subtraction.

  3. Innate Immunity to Adenovirus

    PubMed Central

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka

    2014-01-01

    Abstract Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate–adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features. PMID:24512150

  4. Innate immunity to adenovirus.

    PubMed

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka; Greber, Urs F

    2014-04-01

    Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate-adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features.

  5. Adenovirus structure.

    PubMed

    Rux, John J; Burnett, Roger M

    2004-12-01

    Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

  6. Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method

    PubMed Central

    2011-01-01

    Conventional parenteral injection of vaccines is limited in its ability to induce locally-produced immune responses in the respiratory tract, and has logistical disadvantages in widespread vaccine administration. Recent studies suggest that intranasal delivery or vaccination in the respiratory tract with recombinant viral vectors can enhance immunogenicity and protection against respiratory diseases such as influenza and tuberculosis, and can offer more broad-based generalized protection by eliciting durable mucosal immune responses. Controlled aerosolization is a method to minimize vaccine particle size and ensure delivery to the lower respiratory tract. Here, we characterize the dynamics of aerosolization and show the effects of vaccine concentration on particle size, vector viability, and the actual delivered dose of an aerosolized adenoviral vector. In addition, we demonstrate that aerosol delivery of a recombinant adenoviral vaccine encoding H1N1 hemagglutinin is immunogenic and protects ferrets against homologous viral challenge. Overall, aerosol delivery offers comparable protection to intramuscular injection, and represents an attractive vaccine delivery method for broad-based immunization campaigns. PMID:22103776

  7. 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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of

  8. 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.

    PubMed

    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

  9. Adenovirus vector carrying REIC/DKK-3 gene: neoadjuvant intraprostatic injection for high-risk localized prostate cancer undergoing radical prostatectomy

    PubMed Central

    Kumon, H; Ariyoshi, Y; Sasaki, K; Sadahira, T; Araki, M; Ebara, S; Yanai, H; Watanabe, M; Nasu, Y

    2016-01-01

    As the First-In-Human study of in situ gene therapy using an adenovirus vector carrying the human REIC (reduced expression in immortalized cell)/Dkk-3 gene (Ad-REIC), we conducted neoadjuvant intraprostatic injections in patients with high-risk localized prostate cancer undergoing radical prostatectomy (RP). Patients with recurrence probability of 35% or more within 5 years following RP, as calculated by Kattan's nomogram, were enrolled. Patients received two ultrasound-guided intratumoral injections at 2-week intervals, followed by RP 6 weeks after the second injection. After confirming the safety of the therapeutic interventions with initially planned three escalating doses of 1.0 × 1010, 1.0 × 1011 and 1.0 × 1012 viral particles (vp) in 1.0–1.2 ml (n=3, 3 and 6), an additional higher dose of 3.0 × 1012 vp in 3.6 ml (n=6) was further studied. All four DLs including the additional dose level-4 (DL-4) were feasible with no adverse events, except for grade 1 or 2 transient fever. Laboratory toxicities were grade 1 or 2 elevated aspartate transaminase/alanine transaminase (n=4). Regarding antitumor activities, cytopathic effects (tumor degeneration with cytolysis and pyknosis) and remarkable tumor-infiltrating lymphocytes in the targeted tumor areas were detected in a clear dose-dependent manner. Consequently, biochemical recurrence-free survival in DL-4 was significantly more favorable than in patient groups DL-1+2+3. PMID:27767086

  10. Contrasting Effects of Human, Canine, and Hybrid Adenovirus Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Clinical Efficacy▿

    PubMed Central

    Perreau, Matthieu; Mennechet, Franck; Serratrice, Nicolas; Glasgow, Joel N.; Curiel, David T.; Wodrich, Harald; Kremer, Eric J.

    2007-01-01

    Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis of Adenoviridae immunogenicity and that each may have specific clinical advantages. PMID:17229706

  11. Vasculature-Specific Adenovirus Vectors for Gene Therapy of Prostate Cancer

    DTIC Science & Technology

    2008-02-01

    University of Zurich, Switzerland) on developing designed ankyrin repeat protein ligands (DARPins) specific for Ad fiber knob domain, which will be used as...facilitate the identification of the human tumor xenografts grown in mice and also to allow for non- invasive monitoring of tumor growth , we made a...ψ NeoR PCMV hRluc-EGFP Figure 6. Schematic representation of LhRLuc-EGFP genome. LTR – long terminal repeat , ψ - packaging signal, NeoR – G418

  12. Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice.

    PubMed

    Gabaglia, Claudia Raja; DeLaney, Alexandra; Gee, Jennifer; Halder, Ramesh; Graham, Frank L; Gauldie, Jack; Sercarz, Eli E; Braciak, Todd A

    2010-10-14

    Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.

  13. Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice

    PubMed Central

    2010-01-01

    Background Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. Methods The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. Results Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. Conclusion Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment. PMID:20946663

  14. Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

    PubMed Central

    Bagaev, A. V.; Pichugin, A. V.; Lebedeva, E. S.; Lysenko, A. A.; Shmarov, M. M.; Logunov, D. Yu.; Naroditsky, B. S.; Ataullakhanov, R. I.; Khaitov, R. M.; Gintsburg, A. L.

    2014-01-01

    Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling. PMID:25558392

  15. Adenovirus DNA replication in vitro: site-directed mutagenesis of the nuclear factor I binding site of the Ad2 origin.

    PubMed Central

    de Vries, E; van Driel, W; Tromp, M; van Boom, J; van der Vliet, P C

    1985-01-01

    The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I. Images PMID:4040630

  16. The adenovirus type 2-simian virus 40 hybrid virus Ad2+ND4 requires deletion variants to grow in monkey cells.

    PubMed Central

    Lewis, A M; Westphal, H

    1983-01-01

    The Ad2+ND4 virus is an adenovirus type 2 (Ad2)-simian virus 40 (SV40) recombination. The Ad2 genome of this recombinant has a rearrangement within early region 3; Ad2 DNA sequences between map positions 81.3 and 85.5 have been deleted, and the SV40 DNA sequences between map positions 0.11 and 0.626 have been inserted into the deletion in an 81.3-0.626 orientation. Nonhybrid Ad2 is defective in monkey cells; however, the Ad2+ND4 virus can replicate in monkey cells due to the expression of the SV40-enhancing function encoded by the DNA insert. Stocks of the Ad2+ND4 hybrid were produced in primary monkey cells by using the progeny of a three-step plaque purification procedure and were considered to be homogeneous populations of Ad2+ND4 virions because they induced plaques in primary monkey cells by first-order kinetics. By studying the kinetics of plaque induction in continuous lines (BSC-1 and CV-1) of monkey cells, we have found that stocks (prepared with virions before and after plaque purification) of Ad2+ND4 are actually heterogeneous populations of Ad2+ND4 virions and Ad2+ND4 deletion variants that lack SV40 and frequently Ad2 DNA sequences at the left Ad2-SV40 junction. Due to the defectiveness of the Ad2+ND4 virus, the production of progeny in BSC-1 and CV-1 cells requires complementation between the Ad2+ND4 genome and the genome of an Ad2+ND4 deletion variant. Since the deletion variants that have been obtained from Ad2+ND4 stocks do not express the SV40-enhancing function in that they cannot produce progeny in monkey cells, we conclude that they are providing an Ad2 component that is essential for the production of Ad2+ND4 progeny. These data imply that the Ad2+ND4 virus is incapable of replicating in singly infected primary monkey cells without generating deletion variants that are missing various amounts of DNA around the left Ad2-SV40 junction in the hybrid genome. As the deletion variants that arise from the Ad2+ND4 virus are created by nonhomologous

  17. Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

    PubMed

    Zaiss, Anne K; Vilaysane, Akosua; Cotter, Matthew J; Clark, Sharon A; Meijndert, H Christopher; Colarusso, Pina; Yates, Robin M; Petrilli, Virginie; Tschopp, Jurg; Muruve, Daniel A

    2009-06-01

    Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

  18. Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus

    SciTech Connect

    Qi, Yanxin; Guo, Huanhuan; Hu, Ningning; He, Dongyun; Zhang, Shi; Chu, Yunjie; Huang, Yubin; Li, Xiao; Sun, LiLi; Jin, Ningyi

    2014-10-15

    Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose > 5 × 10{sup 10} VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25 × 10{sup 10} VP/kg) and beagles (2.5 × 10{sup 9} VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35 × 10{sup 10} VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67 × 10{sup 8} VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. - Highlights: • We use the rodents and non-rodents animal models to evaluation Ad-hTERT-E1a-Apoptin. • Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. • Demonstrate the safety and feasibility dose of injected Ad

  19. General Strategy for Broadening Adenovirus Tropism

    PubMed Central

    Fontana, Laura; Nuzzo, Maurizio; Urbanelli, Lorena; Monaci, Paolo

    2003-01-01

    In spite of its broad host range, adenovirus type 5 (Ad5) transduces a number of clinically relevant tissues and cell types inefficiently, mostly because of low expression of the coxsackievirus-adenovirus receptor (CAR). To improve gene transfer to such cells, we modified the Ad5 fiber knob to recognize novel receptors. We expressed a functional Ad5 fiber knob domain on the capsid of phage λ and employed this display system to construct a large collection of ligands in the HI loop of the Ad5 knob. Panning this library on the CAR-negative mouse fibroblast cell line NIH 3T3 resulted in the identification of three clones with increased binding to these cells. Adenoviruses incorporating these ligands in the fiber gene transduced NIH 3T3 cells 2 or 3 orders of magnitude better than the parent vector. The same nonnative tropism was revealed in other cell types, independently of CAR expression. These Ad5 derivatives proved capable of transducing mouse and human primary immature dendritic cells with up to 100-fold increased efficiency. PMID:14512557

  20. Crystal Structure of the Fibre Head Domain of the Atadenovirus Snake Adenovirus 1

    PubMed Central

    Singh, Abhimanyu K.; Menéndez-Conejero, Rosa; San Martín, Carmen; van Raaij, Mark J.

    2014-01-01

    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. PMID:25486282

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

    PubMed

    Singh, Abhimanyu K; Menéndez-Conejero, Rosa; San Martín, Carmen; van Raaij, Mark J

    2014-01-01

    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.

  2. Polymeric oncolytic adenovirus for cancer gene therapy

    PubMed Central

    Choi, Joung-Woo; Lee, Young Sook; Yun, Chae-Ok; Kim, Sung Wan

    2015-01-01

    Oncolytic adenovirus (Ad) vectors present a promising modality to treat cancer. Many clinical trials have been done with either naked oncolytic Ad or combination with chemotherapies. However, the systemic injection of oncolytic Ad in clinical applications is restricted due to significant liver toxicity and immunogenicity. To overcome these issues, Ad has been engineered physically or chemically with numerous polymers for shielding the Ad surface, accomplishing extended blood circulation time and reduced immunogenicity as well as hepatotoxicity. In this review, we describe and classify the characteristics of polymer modified oncolytic Ad following each strategy for cancer treatment. Furthermore, this review concludes with the highlights of various polymer-coated Ads and their prospects, and directions for future research. PMID:26453806

  3. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

    PubMed

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV.

  4. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    PubMed Central

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211

  5. Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus.

    PubMed

    Qi, Yanxin; Guo, Huanhuan; Hu, Ningning; He, Dongyun; Zhang, Shi; Chu, Yunjie; Huang, Yubin; Li, Xiao; Sun, LiLi; Jin, Ningyi

    2014-10-15

    Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0×10(8), 2.5×10(9), and 1.25×10(10) viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0×10(8), 2.5×10(9), and 1.25×10(10) VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose>5×10(10) VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25×10(10) VP/kg) and beagles (2.5×10(9) VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35×10(10) VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67×10(8) VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    SciTech Connect

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

    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.

  7. Rescue administration of a helper-dependent adenovirus vector with long-term efficacy in dogs with glycogen storage disease type Ia.

    PubMed

    Crane, B; Luo, X; Demaster, A; Williams, K D; Kozink, D M; Zhang, P; Brown, T T; Pinto, C R; Oka, K; Sun, F; Jackson, M W; Chan, L; Koeberl, D D

    2012-04-01

    Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.

  8. Use of replication-defective adenoviruses to develop vaccines and biotherapeutics against foot-and-mouth disease

    USDA-ARS?s Scientific Manuscript database

    We have developed a replication-defective human adenovirus (Ad5) vectored foot-and-mouth disease (FMD) vaccine platform that with one inoculation protects both swine and cattle from subsequent challenge with homologous virus. This Ad5-FMD vaccine delivery system has undergone testing following the r...

  9. A rotating hairy AdS3 black hole with the metric having only one Killing vector field

    NASA Astrophysics Data System (ADS)

    Iizuka, Norihiro; Ishibashi, Akihiro; Maeda, Kengo

    2015-08-01

    We perturbatively construct a three-dimensional rotating AdS black hole with a real scalar hair. We choose the mass of a scalar field slightly above the Breitenlohner-Freedman bound and impose a general boundary condition for the bulk scalar field at AdS infinity. We first show that rotating BTZ black holes are unstable against scalar field perturbations under our more general boundary condition. Next we construct a rotating hairy black hole perturbatively with respect to a small amplitude ɛ of the scalar field, up to O( ɛ 4). Our hairy black hole is stationary and exhibits no dissipation, but the lumps of the non-linearly perturbed geometry break axial symmetry, thus providing the first example of a rotating black hole whose metric admits only one Killing vector field. Furthermore, we numerically show that the entropy of our hairy black hole is larger than that of the BTZ black hole with the same energy and the angular momentum. We briefly discuss if our rotating hairy black hole in lumpy geometry could be the endpoint of the instability.

  10. Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

    PubMed Central

    Jouneau, Luc; Bourge, Mickael; Bouet-Cararo, Coraline; Bonneau, Michel; Zientara, Stephan; Klonjkowski, Bernard; Schwartz-Cornil, Isabelle

    2012-01-01

    Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b+ -type and CD103+ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b+ -type DCs was far higher and broader than in the CD103+ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b+ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b+ DC type is more responsive to CAV2 than the CD103+ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness. PMID:23300693

  11. [Effect of recombinant human p53 adenovirus (Ad-p53) combined with EGFR inhibitor gefitinib on the sensitivity of breast cancer MDA-MB-468 cells].

    PubMed

    Wang, Xinzhao; Guan, Xiyun; Wang, Leilei; Xie, Li; Liu, Qi; Yu, Zhiyong

    2014-12-01

    To observe the impact of concurrent administration of recombinant human p53 adenovirus (Ad-p53) with EGFR inhibitor gefitinib on breast cancer MDA-MB-468 cells. MDA-MB-468 cells were treated with Ad-p53 and/or gefitinib. The effect of Ad-p53 and gefitinib on the growth of MDA-MB-468 cells was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to detect the alteration of p53,EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and apoptosis-related proteins. Ad-p53 combined with gefitinib was used in vivo to explore their effect on tumor xenograft in nude mice. Immunohistochemistry was used to detect the p53 expression in vivo. The MTT assay showed a stronger inhibitory effect of gefitinib on MDA-MB-468 cells infected with Ad-p53 than on the control cells. Cell apoptosis assay revealed that the apoptosis rates of MDA-MB-468 cells in vehicle-treated group, Ad-p53 group, gefitinib group, and combination group were 8.5%, 17.4%, 20.5% and 32.6%, respectively. The apoptosis rate of MDA-MB-468 cells in the combination group was higher than that in other groups (P < 0.05, for all) . Western blot analysis revealed that the expression of p53 was significantly up-regulated in the presence of Ad-p53. The combination of Ad-p53 and gefitinib significantly down-regulated p-Akt (S473)(P < 0.01) and up-regulated caspase-9 and cleaved caspase-3 (P < 0.01 for both). Tumor inhibition rates (TIR) in the Ad-p53, gefitinib, and combination groups were 35.7%, 28.7% and 74.4%, respectively. Ad-p53 and gefitinib combination therapy significantly reduced the tumor burden in nude mice (P < 0.05 for all).Immunohistochemistry showed that after intratumoral administration of Ad-p53, wild-type p53 was increased (P < 0.01). p53 expressions in the vehicle-treated, Ad-p53, gefitinib and combination groups were 45.2%, 80.1%, 50.7% and 90.6%, respectively. Wild-type p53 may reverse the sensitivity of MDA-MB-468 cells to gefitinib through

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

    PubMed

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes; Bassi, Maria Rosaria; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2012-01-01

    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.

  13. Requirements for Receptor Engagement during Infection by Adenovirus Complexed with Blood Coagulation Factor X

    PubMed Central

    Bradshaw, Angela C.; Parker, Alan L.; Duffy, Margaret R.; Coughlan, Lynda; van Rooijen, Nico; Kähäri, Veli-Matti; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define

  14. Gauge theories on A(dS) space and Killing vectors

    SciTech Connect

    Banerjee, Rabin Majhi, Bibhas Ranjan

    2008-03-15

    We provide a general technique for collectively analysing a manifestly covariant formulation of non-abelian gauge theories on both anti-de Sitter as well as de Sitter spaces. This is done by stereographically projecting the corresponding theories, defined on a flat Minkowski space, onto the surface of the A(dS) hyperboloid. The gauge and matter fields in the two descriptions are mapped by conformal Killing vectors and conformal Killing spinors, respectively. A bilinear map connecting the spinors with the vector is established. Different forms of gauge fixing conditions and their equivalence are discussed. The U(1) axial anomaly as well as the non-abelian covariant and consistent chiral anomalies on A(dS) space are obtained. Electric-magnetic duality is demonstrated. The zero curvature limit is shown to yield consistent findings.

  15. Full vector archaeomagnetic data and Bayesian modelling for 1300 to 1750 AD

    NASA Astrophysics Data System (ADS)

    Schnepp, E.; Lanos, P.; Chauvin, A.

    2009-04-01

    The data base of geomagnetic palaeointensities obtained from archaeological artefacts is poor and very scattered for Western and Central Europe. High precision palaeointensities have been determined from a single archaeological site in Lübeck (Germany) where a sequence of 25 bread-oven-floors has been preserved in a bakery from medieval times until today. Age dating confines the time interval from about 1300 AD to about 1750 AD. Palaeomagnetic directions have been determined from each oven-floor (Schnepp et al., JGR, 2003). Palaeointensity was measured from selected specimens with the double-heating Thellier method and reliable palaeointensity results have been obtained. Tests for thermoremanent magnetisation anisotropy have been performed, but did not show a significant change, while a cooling rate correction was not necessary. 22 mean palaeointensity values derived from the oven-floors show maxima in the 15th and early 17th century AD, followed by a decrease of palaeointensity of about 25% until 1750 AD. The Thellier experiments provided also new characteristic remanent magnetisation directions which were included in the data set. Mean directions have been recalculated. Palaeointensity together with the directions represent a record of about 450 years full vector secular variation. From this full vector data set a secular variation curve has been calculated using a Bayesian modelling taking dating errors, all errors on the field vector and stratigraphy into account. A smooth curve with an error envelope was obtained which compares very well with the gufm1 geomagnetic model (Jackson et al., Phil. Trans. R. Soc. Lond. A, 2000) obtained from historical observations starting at 1600 AD. Comparison of the marginal curve obtained for palaeointensity with a selected data set of archaeomagnetic intensities from Western and Central Europe will be discussed.

  16. RGD Inclusion in the Hexon Monomer Provides Adenovirus Type 5-Based Vectors with a Fiber Knob-Independent Pathway for Infection

    PubMed Central

    Vigne, Emmanuelle; Mahfouz, Irene; Dedieu, Jean-Francois; Brie, Anne; Perricaudet, Michel; Yeh, Patrice

    1999-01-01

    Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine “isogenic” viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An αv-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands. PMID:10233980

  17. A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

    PubMed

    Karen, Kasey A; Deal, Cailin; Adams, Robert J; Nielsen, Carolyn; Ward, Cameron; Espinosa, Diego A; Xie, Jane; Zavala, Fidel; Ketner, Gary

    2015-01-01

    Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.

  18. Enhanced Peptide of Prostate Cancer Using Targeted Adenoviral Vectors

    DTIC Science & Technology

    2005-06-01

    greater than that observed in tumors injected with control adenovirus (1.4 - 1..6% ID/g). Another adenovirus encoding for both SSTR2 and cytosine deaminase ...for treating prostate cancer xenografts which involves the use of an adenoviral vector encoding for both SSTR2 and the cytosine deaminase (CD) enzyme...SSTR2 and bacterial cytosine deaminase (CD) was performed in a manner similar to that previously described. The AdEasy system was used to generate the

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

    PubMed

    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.

  20. Identification of Novel Inverted Terminal Repeat (ITR) Deletions of Human Adenovirus (AD) From Infected Host: Virulent Ads Containing Mixed Populations of Genomic Sequences

    DTIC Science & Technology

    2006-11-01

    basepairs) in size (Houng, et al., 2006; Shinagawa , M et al 1980; Shinagawa , M et al 1982). The representative ITRs for human Ad serotypes from...chemical degradation procedures of Maxam and Gilbert ( Shinagawa et al., 1980; Stillman et al, 1982; Tokunaga et al., 1982). A simple DNA sequencing...recruits: Epidemiologic and immunologic risk factors in healthy, young adults. J Med Virol 65:710-718. 16. Shinagawa , M., and R. Padmanabhan. 1980

  1. Increased therapeutic efficacy of the prostate-specific oncolytic adenovirus Ad[I/PPT-E1A] by reduction of the insulator size and introduction of the full-length E3 region.

    PubMed

    Danielsson, A; Dzojic, H; Nilsson, B; Essand, M

    2008-04-01

    Conditionally replicating adenoviruses are developing as a complement to traditional cancer therapies. Ad[I/PPT-E1A] is an E1B/E3-deleted virus that replicates exclusively in prostate cells, since the expression of E1A is controlled by the recombinant 1.4 kb prostate-specific PPT promoter. The transcriptional integrity of PPT is maintained by the 3.0 kb mouse H19 insulator that was introduced directly upstream of the PPT sequence. In order to increase the cloning capacity to be able to reintroduce E3 sequences in the 35.7 kb Ad[I/PPT-E1A] genome, various shorter insulators were examined in a luciferase reporter gene assay. It was found that the 1.6 kb core H19 insulator (i) improves the activity of PPT, compared to the 3.0 kb full-length insulator, while still maintaining prostate cell specificity and releasing 1.4 kb of space for insertion of additional sequences. To improve the ability of the virus to efficiently lyse infected cells and persist in vivo, we inserted the adenovirus death protein (ADP) or the full-length adenovirus E3 region. The oncolytic activity of PPT-E1A-based viruses was studied using MTS, crystal violet and replication assays. The virus with the reintroduced full-length E3-region (Ad[i/PPT-E1A, E3]) showed the highest cytopathic effects in vitro. Furthermore, this virus suppressed the growth of aggressively growing prostate tumors in vivo. Therefore, we conclude that Ad[i/PPT-E1A, E3] is a prostate-specific oncolytic adenovirus with a high potential for treating localized prostate cancer.

  2. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    PubMed

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-06-02

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.

  3. [Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)].

    PubMed

    Logunov, D Iu; Cherenova, L V; Shmarov, M M; Shashkova, E V; Verkhovskaia, L V; Doronin, K K; Naroditskiĭ, B S

    2002-01-01

    Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

  4. Directed adenovirus evolution using engineered mutator viral polymerases

    PubMed Central

    Uil, Taco G.; Vellinga, Jort; de Vrij, Jeroen; van den Hengel, Sanne K.; Rabelink, Martijn J. W. E.; Cramer, Steve J.; Eekels, Julia J. M.; Ariyurek, Yavuz; van Galen, Michiel; Hoeben, Rob C.

    2011-01-01

    Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing. PMID:21138963

  5. Advances and Future Challenges in Adenoviral Vector Pharmacology and Targeting

    PubMed Central

    Khare, Reeti; Chen, Christopher Y; Weaver, Eric A; Barry, Michael A

    2011-01-01

    Adenovirus is a robust vector for therapeutic applications, but its use is limited by our understanding of its complex in vivo pharmacology. In this review we describe the necessity of identifying its natural, widespread, and multifaceted interactions with the host since this information will be crucial for efficiently redirecting virus into target cells. In the rational design of vectors, the notion of overcoming a sequence of viral “sinks” must be combined with re-targeting to target populations with capsid as well as shielding the vectors from pre-existing or toxic immune responses. It must also be noted that most known adenoviral pharmacology is deduced from the most commonly used serotypes, Ad5 and Ad2. However, these serotypes may not represent all adenoviruses, and may not even represent the most useful vectors for all purposes. Chimeras between Ad serotypes may become useful in engineering vectors that can selectively evade substantial viral traps, such as Kupffer cells, while retaining the robust qualities of Ad5. Similarly, vectorizing other Ad serotypes may become useful in avoiding immunity against Ad5 altogether. Taken together, this research on basic adenovirus biology will be necessary in developing vectors that interact more strategically with the host for the most optimal therapeutic effect. PMID:21453281

  6. Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

    PubMed Central

    2013-01-01

    Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer. PMID:23937994

  7. Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice

    PubMed Central

    Liu, Shan; Jackson, Andrew; Beloor, Jagadish; Kumar, Priti; Sutton, Richard E.

    2015-01-01

    Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4+ T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans. PMID:25953321

  8. Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

    PubMed Central

    Bradshaw, Angela C.; Coughlan, Lynda; Miller, Ashley M.; Alba, Raul; van Rooijen, Nico; Nicklin, Stuart A.; Baker, Andrew H.

    2012-01-01

    The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton–αvintegrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1 + sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery. PMID:22626939

  9. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

    PubMed Central

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M.; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D.; Townes, Tim M.

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  10. Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials.

    PubMed

    Huang, Yunda; Follmann, Dean; Nason, Martha; Zhang, Lily; Huang, Ying; Mehrotra, Devan V; Moodie, Zoe; Metch, Barbara; Janes, Holly; Keefer, Michael C; Churchyard, Gavin; Robb, Merlin L; Fast, Patricia E; Duerr, Ann; McElrath, M Juliana; Corey, Lawrence; Mascola, John R; Graham, Barney S; Sobieszczyk, Magdalena E; Kublin, James G; Robertson, Michael; Hammer, Scott M; Gray, Glenda E; Buchbinder, Susan P; Gilbert, Peter B

    2015-01-01

    Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow-up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines. We included participant-level data from all three efficacy trials, and three Phase 1-2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests. Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99-1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11-1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61-1.26, P = 0.48). Results were similar when including the Phase 1-2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5 vaccine-increased risk was concentrated in

  11. Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials

    PubMed Central

    Huang, Yunda; Follmann, Dean; Nason, Martha; Zhang, Lily; Huang, Ying; Mehrotra, Devan V.; Moodie, Zoe; Metch, Barbara; Janes, Holly; Keefer, Michael C.; Churchyard, Gavin; Robb, Merlin L.; Fast, Patricia E.; Duerr, Ann; McElrath, M. Juliana; Corey, Lawrence; Mascola, John R.; Graham, Barney S.; Sobieszczyk, Magdalena E.; Kublin, James G.; Robertson, Michael; Hammer, Scott M.; Gray, Glenda E.; Buchbinder, Susan P.; Gilbert, Peter B.

    2015-01-01

    Background Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow–up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines. Methods We included participant-level data from all three efficacy trials, and three Phase 1–2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests. Findings Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99–1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11–1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61–1.26, P = 0.48). Results were similar when including the Phase 1–2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5

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

    PubMed Central

    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

  13. Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea.

    PubMed

    Austin, Bobbie Ann; Halford, William P; Williams, Bryan R G; Carr, Daniel J J

    2007-04-15

    An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.

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

    PubMed

    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.

  15. 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.

    PubMed

    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

  16. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

    Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

  17. STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    PubMed Central

    Spurrell, Emma; Gangeswaran, Rathi; Wang, Pengju; Cao, Fengyu; Gao, Dongling; Feng, Baisui; Wold, William; Tollefson, Ann

    2014-01-01

    Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy. PMID:24335311

  18. Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus.

    PubMed

    Nguyen, Thanh H; Vidovszky, Márton Z; Ballmann, Mónika Z; Sanz-Gaitero, Marta; Singh, Abhimanyu K; Harrach, Balázs; Benkő, Mária; van Raaij, Mark J

    2015-05-22

    In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus. We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414-535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution. Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed. Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of

  19. Simultaneous CAR- and alpha V integrin-binding ablation fails to reduce Ad5 liver tropism.

    PubMed

    Martin, Karine; Brie, Anne; Saulnier, Patrick; Perricaudet, Michel; Yeh, Patrice; Vigne, Emmanuelle

    2003-09-01

    Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and alpha(V) integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and alpha(V) integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.

  20. Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy

    PubMed Central

    Farzad, Lisa; Cerullo, Vincenzo; Yagyu, Shigeki; Bertin, Terry; Hemminki, Akseli; Rooney, Cliona; Lee, Brendan; Suzuki, Masataka

    2014-01-01

    Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy. PMID:27119096

  1. The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

    PubMed Central

    Pantelidou, Constantia; Cherubini, Gioia; Lemoine, Nick R.; Halldén, Gunnel

    2016-01-01

    Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics. PMID:26872382

  2. Adenovirus Vector-Induced CD8+ T Effector Memory Cell Differentiation and Recirculation, But Not Proliferation, Are Important for Protective Immunity Against Experimental Trypanosoma cruzi Infection

    PubMed Central

    Vasconcelos, José Ronnie; Dominguez, Mariana R.; Neves, Ramon L.; Ersching, Jonatan; Araújo, Adriano; Santos, Luara I.; Virgilio, Fernando S.; Machado, Alexandre V.; Bruna-Romero, Oscar; Gazzinelli, Ricardo T.

    2014-01-01

    Abstract Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8+ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8+ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1High CD27Low CD43Low CD183LowT-betHigh EomesLow phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8+ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8+ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8+ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity. PMID:24568548

  3. Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge.

    PubMed

    Schutta, Christopher; Barrera, José; Pisano, Melia; Zsak, Laszlo; Grubman, Marvin J; Mayr, Gregory A; Moraes, Mauro P; Kamicker, Barbara J; Brake, David A; Ettyreddy, Damodar; Brough, Douglas E; Butman, Bryan T; Neilan, John G

    2016-06-08

    The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

  4. Fiber-modified adenovirus for central nervous system Parkinson's disease gene therapy.

    PubMed

    Lewis, Travis B; Glasgow, Joel N; Harms, Ashley S; Standaert, David G; Curiel, David T

    2014-08-21

    Gene-based therapies for neurological diseases continue to develop briskly. As disease mechanisms are elucidated, flexible gene delivery platforms incorporating transcriptional regulatory elements, therapeutic genes and targeted delivery are required for the safety and efficacy of these approaches. Adenovirus serotype 5 (Ad5)-based vectors can carry large genetic payloads to provide this flexibility, but do not transduce neuronal cells efficiently. To address this, we have developed a tropism-modified Ad5 vector with neuron-selective targeting properties for evaluation in models of Parkinson disease therapy. A panel of tropism-modified Ad5 vectors was screened for enhanced gene delivery in a neuroblastoma cell line model system. We used these observations to design and construct an unbiased Ad vector platform, consisting of an unmodified Ad5 and a tropism-modified Ad5 vector containing the fiber knob domain from canine Ad serotype 2 (Ad5-CGW-CK2). Delivery to the substantia nigra or striatum showed that this vector produced a neuronally-restricted pattern of gene expression. Many of the transduced neurons were from regions with afferent projections to the injection site, implicating that the vector binds the presynaptic terminal resulting in presynaptic transduction. We show that Ad5-CGW-CK2 can selectively transduce neurons in the brain and hypothesize that this modular platform is potentially adaptable to clinical use.

  5. Conserved Fiber-Penton Base Interaction Revealed by Nearly Atomic Resolution Cryo-Electron Microscopy of the Structure of Adenovirus Provides Insight into Receptor Interaction

    PubMed Central

    Cao, Changchun; Dong, Xiaoyan; Wu, Xiaobing; Wen, Boyun; Ji, Gang

    2012-01-01

    Adenovirus (Ad) cell attachment is initiated by the attachment of the fiber protein to a primary receptor (usually CAR or CD46). This event is followed by the engagement of the penton base protein with a secondary receptor (integrin) via its loop region, which contains an Arg-Gly-Asp (RGD) motif, to trigger virus internalization. To understand the well-orchestrated adenovirus cell attachment process that involves the fiber and the penton base, we reconstructed the structure of an Ad5F35 capsid, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber, at a resolution of approximately 4.2 Å. The fiber-penton base interaction in the cryo-electron microscopic (cryo-EM) structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5, indicating that the fiber-penton base interaction of adenovirus is conserved. Our structure also confirms that the C-terminal segment of the fiber tail domain constitutes the bottom trunk of the fiber shaft. Based on the conserved fiber-penton base interaction, we have proposed a model for the interaction of Ad5F35 with its primary and secondary receptors. This model could provide insight for designing adenovirus gene delivery vectors. PMID:22951835

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Capsid-Incorporation of Antigens into Adenovirus Capsid Proteins for a Vaccine Approach

    PubMed Central

    Matthews, Qiana L.

    2010-01-01

    Some viral vectors are potent inducers of cellular and humoral responses; therefore, viral vectors can be used to vaccinate against cancer or infectious diseases. This report will focus on adenovirus (Ad)-based vectors. Traditional viral-vector vaccination embodies the concept that the vector uses the host-cell machinery to express antigens that are encoded as transgenes within the viral vector. Several preclinical successes have used this approach in animal model systems. However, in some instances, these conventional Ad-based vaccines have yielded suboptimal clinical results. These suboptimal results are ascribed, in part, to preexisting Ad serotype 5 (Ad5) immunity. To address this issue, the “antigen capsid-incorporation” strategy has been developed to circumvent the drawbacks associated with conventional transgene expression of antigens by Ad vectors. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. Incorporating immunogenic peptides into the Ad capsid offers potential advantages. Importantly, vaccination by means of the antigen capsid-incorporated approach results in a strong humoral response, similar to the response generated by native Ad capsid proteins. This strategy also allows for the boosting of antigenic specific responses. This strategy may be the way forward for improved vaccine schemes, especially for those infections requiring a strong humoral antigenic response. PMID:21047139

  8. Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine

    PubMed Central

    Tran, Kaylie N.; Croyle, Maria A.; Strong, James E.; Feldmann, Heinz; Kobinger, Gary P.

    2009-01-01

    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 previous generation

  9. Immune Responses to Adenovirus and Adeno-Associated Vectors Used for Gene Therapy of Brain Diseases: The Role of Immunological Synapses in Understanding the Cell Biology of Neuroimmune Interactions

    PubMed Central

    Lowenstein, Pedro R.; Mandel, Ronald J.; Xiong, Wei-dong; Kroeger, Kurt; Castro, Maria G.

    2008-01-01

    Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint. “The world does not speak. Only we do.” Richard Rorty, Contingency, irony, and solidarity, 1989. PMID:17979681

  10. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  11. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases

    PubMed Central

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R.; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B.; Iozzo, Renato V.; Seth, Prem

    2015-01-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases. PMID:26467629

  12. Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression.

    PubMed

    Lee, Cho-Hee; Kasala, Dayananda; Na, Youjin; Lee, Min Sang; Kim, Sung Wan; Jeong, Ji Hoon; Yun, Chae-Ok

    2014-07-01

    Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

  13. Cell-specific induction of sensitivity to ganciclovir in medullary thyroid carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase.

    PubMed

    Minemura, K; Takeda, T; Minemura, K; Nagasawa, T; Zhang, R; Leopardi, R; DeGroot, L J

    2000-05-01

    Herpes simplex virus thymidine kinase (HSVtk) gene transfer followed by ganciclovir administration is a common strategy for experimental cancer therapy. To evaluate the feasibility of using the human calcitonin promoter to target medullary thyroid carcinoma (MTC), we developed adenovirus vectors containing Escherichia coli beta-galactosidase gene under the control of the CALC-I promoter (AdCTlacZ), or the human cytomegalovirus promoter (AdCMVlacZ). Beta-galactosidase activity driven by the CALC-I promoter was higher than by the CMV promoter in rat MTC cells after infection with adenovirus vectors. AdCTlacZ induced an equal or lower expression level of beta-galactosidase in TT (human MTC), T98G, Cos1, HepG2, and HeLa cells compared with AdCMVlacZ. To inhibit the growth of MTC cells, we developed two adenovirus vectors, AdCMVtk carrying HSVtk driven by the cytomegalovirus promoter and AdDCTtk containing a human CALC-I minigene under the control of the CALC-I promoter. HSVtk is fused to a portion of calcitonin coded in exon 4 to direct cell-specific regulation of splicing. All cell lines infected with AdCMVtk were rendered sensitive to ganciclovir, whereas T98G and Cos1 cells infected with AdDCTtk were not affected. Cell killing was also observed in HeLa, HepG2, rat MTC and TT cells infected with AdDCTtk.

  14. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.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 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. 29 figs.

  15. Activated recombinant adenovirus proteinases

    DOEpatents

    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.

  16. Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents

    SciTech Connect

    Shashkova, Elena V.; May, Shannon M.; Barry, Michael A.

    2009-11-25

    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.

  17. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells*

    PubMed Central

    Luo, Xu-wei; Liu, Kang; Chen, Zhu; Zhao, Ming; Han, Xiao-wei; Bai, Yi-guang; Feng, Gang

    2016-01-01

    Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells. PMID:26739524

  18. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

    PubMed

    Lee, Choon-Taek; Park, Kyung-Ho; Yanagisawa, Kiyoshi; Adachi, Yasushi; Ohm, Joyce E; Nadaf, Sorena; Dikov, Mikhail M; Curiel, David T; Carbone, David P

    2004-09-15

    Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

  19. Nanoporous Crystals of Chicken Embryo Lethal Orphan (CELO) Adenovirus Major Coat Protein, Hexon

    PubMed Central

    Xu, Lan; Benson, Stacy D.; Burnett, Roger M.

    2007-01-01

    CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 Å resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 (a = b = 157.8 Å, c = 114.2 Å, γ = 120º) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large ~100 Å diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts. PMID:17071105

  20. Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

    PubMed Central

    Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

    1995-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber

  1. Polyethylene glycol modification of adenovirus reduces platelet activation, endothelial cell activation, and thrombocytopenia.

    PubMed

    Hofherr, Sean E; Mok, Hoyin; Gushiken, Francisca C; Lopez, Jose A; Barry, Michael A

    2007-09-01

    Thrombocytopenia is one of the complications for in vivo administration of adenovirus serotype 5 (Ad5) vectors after intravenous injection. In this paper, we investigated the mechanism of Ad5-induced thrombocytopenia and how these effects are attenuated by polyethylene glycol (PEG) modification of Ad5 (Ad-PEG). After intravenous injection, accelerated platelet loss was observed in Ad-injected mice but not in their Ad-PEG-injected counterparts. This platelet loss induced by Ad5 corresponded with increases in coagulation D-dimer levels, splenomegaly, and, later, production of megakaryocytes in the bone marrow. In contrast, these responses were blunted or ablated after injection of Ad-PEG. Ad5 activated both platelets and endothelial cells directly in vitro as evidenced by induction of P-selectin and the formation of von Willebrand factor-platelet strings and in vivo as evidenced by the induction of E-selectin messenger RNA. PEGylation blunted these observed activations. These data suggest that Ad5 may induce thrombocytopenia by direct activation of endothelial cells in addition to its direct effects on platelets. This link provides an important clue for the understanding of the mechanisms of thrombocytopenia associated with Ad5. Given that PEGylation blunted interactions of Ad with platelets and endothelial cells, reduced D-dimer formation, reduced thrombocytopenia, and reduced splenomegaly, these data suggest that this simple vector modification may have utility to improve the safety of Ad vectors for human gene therapy.

  2. Combined transductional untargeting/retargeting and transcriptional restriction enhance adenovirus gene targeting and therapy for hepatic colorectal cancer tumors

    PubMed Central

    Li, Hua-Jung; Everts, Maaike; Yamamoto, Masato; Curiel, David T.; Herschman, Harvey R.

    2009-01-01

    Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors, and other epithelial tumors, often express both cyclooxygenase 2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus vectors infect liver and lack tumor tropism, they cannot be utilized for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional adenovirus hepatic untargeting and tumor retargeting by a bispecific adapter, sCARhMFE, composed of sCAR (the Coxsackie and Adenovirus Receptor ectodomain) and MFE-23 (a single-chain anti-CEA antibody), to untarget liver following intravenous administration of adenovirus vectors expressing firefly luciferase and to retarget virus to hepatic colorectal tumor xenografts and non-small cell lung tumor xenografts. To improve both liver untargeting and tumor retargeting, we developed sCARfMFE, a trimerized sCARhMFE adapter. Trimerization greatly improves both untargeting of CAR-dependent adenovirus infection and CEA-dependent virus retargeting, in culture and in vivo. Combining sCARfMFE bispecific adapter transductional liver untargeting and transductional tumor retargeting with COX-2 transcriptional tumor-restricted transgene expression increases systemically-administered adenovirus therapeutic efficacy for hepatic CRC tumors, using Herpes Virus Type 1 thymidine kinase as a therapeutic gene in conjunction with the prodrug ganciclovir. Both transductional untargeting and COX-2 transcriptional restriction also reduce HSV1-tk/GCV hepatic toxicity. In addition, transductional sCARfMFE untargeting reduces the innate immune response to systemic adenovirus administration. Combined transductional liver Ad untargeting, transductional tumor retargeting and transcriptional transgene restriction suggests a means to engineer practical, effective therapeutic agents for hepatic CRC metastases in particular, as well as hepatic metastases of other epithelial

  3. Chemical Modification with High Molecular Weight Polyethylene Glycol Reduces Transduction of Hepatocytes and Increases Efficacy of Intravenously Delivered Oncolytic Adenovirus

    PubMed Central

    Doronin, Konstantin; Shashkova, Elena V.; May, Shannon M.; Hofherr, Sean E.

    2009-01-01

    Abstract Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites. PMID:19469693

  4. Intratumoral Injection of an Adenovirus Expressing Interleukin 2 Induces Regression and Immunity in a Murine Breast Cancer Model

    NASA Astrophysics Data System (ADS)

    Addison, Christina L.; Braciak, Todd; Ralston, Robert; Muller, William J.; Gauldie, Jack; Graham, Frank L.

    1995-08-01

    Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

  5. Adenoviral vector-based strategies against infectious disease and cancer

    PubMed Central

    Zhang, Chao; Zhou, Dongming

    2016-01-01

    ABSTRACT Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vector-based strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. PMID:27105067

  6. Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector

    PubMed Central

    Bukh, Irene; Calcedo, Roberto; Roy, Soumitra; Carnathan, Diane G.; Grant, Rebecca; Qin, Qiuyue; Boyd, Surina; Ratcliffe, Sarah J.; Veeder, Christin L.; Bellamy, Scarlett L.; Betts, Michael R.

    2014-01-01

    ABSTRACT The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV

  7. Genetic Incorporation of Human Metallothionein into the Adenovirus Protein IX for Non-Invasive SPECT Imaging

    PubMed Central

    Mathis, J. Michael; Bhatia, Shilpa; Khandelwal, Alok; Kovesdi, Imre; Lokitz, Stephen J.; Odaka, Yoshi; Takalkar, Amol M.; Terry, Tracee; Curiel, David T.

    2011-01-01

    As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of 99mTc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a 99mTc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo. PMID:21347423

  8. Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system.

    PubMed

    Wang, Hongzhi; Feng, Qiang; Wei, Lei; Zhuo, Liling; Chen, Hao; Diao, Youxiang; Tang, Yi

    2017-10-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus, which was first isolated in the tropics during the 1970s. Recently, a disease characterized by ovarian haemorrhage and neurological symptoms was observed in ducks in China, which threatens poultry production. However, there is no suitable vaccination strategy or effective antiviral drugs to combat TMUV infections. Consequently, there is an urgent need to develop a new anti-TMUV therapy. In this study, we report an efficient short hairpin RNA (shRNA) delivery strategy for the inhibition of TMUV production using an adenovirus vector system. Using specifically designed shRNAs based on the E and NS5 protein genes of TMUV, the vector-expressed viral genes, TMUV RNA replication and infectious virus production were downregulated at different levels in Vero cells, where the shRNA (NS52) was highly effective in inhibiting TMUV. Using the human adenovirus type 5 shRNA delivery system, the recombinant adenovirus (rAd-NS52) inhibited TMUV multiplication with high efficiency. Furthermore, the significant dose-dependent inhibition of viral RNA copies induced by rAd-NS52 was found in TMUV-infected cells, which could last for at least 96h post infection. Our results indicated that the adenovirus-mediated delivery of shRNAs could play an active role in future TMUV antiviral therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Ad-mTERT-delta19, a conditional replication-competent adenovirus driven by the human telomerase promoter, selectively replicates in and elicits cytopathic effect in a cancer cell-specific manner.

    PubMed

    Kim, Eunhee; Kim, Joo-Hang; Shin, Ha-Youn; Lee, Hansaem; Yang, Jai Myung; Kim, Jungho; Sohn, Joo-Hyuk; Kim, Hoguen; Yun, Chae-Ok

    2003-10-10

    Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, functions to stabilize telomere length during chromosomal replication. Previous studies have shown that hTERT promoter is highly active in most tumor and immortal cell lines but inactive in normal somatic cell types. The use of wild-type hTERT promoter, however, may be limited by its inability to direct high level and cancer cell-specific expression necessary for effective targeted gene therapy. To improve cancer cell specificity and the strength of the hTERT promoter, a modified hTERT, m-hTERT promoter was generated in which additional copies of c-Myc and Sp1 binding sites were incorporated adjacent to the promoter. As assessed using relative lacZ expression, hTERT and m-hTERT promoter activity was significantly upregulated in cancer cells but not in normal cells, and within these upregulated cancer cells, m-hTERT promoter strength was substantially higher than that of the wild-type hTERT. Next, to restrict viral replication to tumor cells, a conditional replication-competent adenoviruses, Ad-TERT-Delta19 and Ad-mTERT-delta19 were generated in which the E1A gene, which is essential for viral replication, was placed under the control of the hTERT and m-hTERT promoter, respectively. While the wild-type Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect only in cancer cells. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. Taken together, present results strongly suggest that the use of the m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of the m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

  10. Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy.

    PubMed

    Choi, Joung-Woo; Park, Ji Won; Na, Youjin; Jung, Soo-Jung; Hwang, June Kyu; Choi, Dongho; Lee, Kyeong Geun; Yun, Chae-Ok

    2015-10-01

    Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy.

  11. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    PubMed

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

  12. Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

    PubMed

    Vragniau, Charles; Hübner, Jens-Martin; Beidler, Peter; Gil, Sucheol; Saydaminova, Kamola; Lu, Zhuo-Zhuang; Yumul, Roma; Wang, Hongjie; Richter, Maximilian; Sova, Pavel; Drescher, Charles; Fender, Pascal; Lieber, André

    2017-03-15

    Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our

  13. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

  14. Adenovirus-Mediated FKHRL1/TM Sensitizes Melanoma Cells to Apoptosis Induced by Temozolomide

    PubMed Central

    Egger, Michael E.; McNally, Lacey R.; Nitz, Jonathan; McMasters, Kelly M.

    2014-01-01

    Abstract Melanoma exhibits variable resistance to the alkylating agent temozolomide (TMZ). We evaluated the potential of adenovirus expressing forkhead human transcription factor like 1 triple mutant (Ad-FKHRL1/TM) to sensitize melanoma cells to TMZ. Four melanoma cell lines were treated with Ad-FKHRL1/TM and TMZ, alone or in combination. Apoptosis was assessed by activation and inhibition of caspase pathway, nuclei fragmentation, and annexin V staining. The potential therapeutic efficacy of Ad-FKHRL1/TM with TMZ was also assessed in a mouse melanoma xenograft model. Combination therapy of Ad-FKHRL1/TM and TMZ resulted in greater cell killing (<20% cell viability) compared with single therapy and controls (p<0.05). Combination indices of Ad-FKHRL1/TM and TMZ therapy indicated significant (p<0.05) synergistic killing effect. Greater apoptosis induction was found in cells treated with Ad-FKHRL1/TM and TMZ than with Ad-FKHRL1/TM or TMZ-treated cells alone. Treatment with TMZ enhanced adenovirus transgene expression in a cell type-dependent manner. In an in vivo model, combination therapy of Ad-FKHRL1/TM with TMZ results in greater tumor growth reduction in comparison with single treatments. We suggest that Ad-FKHRL1/TM is a promising vector to sensitize melanoma cells to TMZ, and that a combination of both approaches would be effective in the clinical setting. PMID:25238278

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

    PubMed

    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.

  16. Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

    PubMed Central

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

    2012-01-01

    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. PMID:22787235

  17. Serological Immunity to Adenovirus serotype 5 is not Associated with Risk of HIV Infection: a Case-control Study

    PubMed Central

    Curlin, Marcel E.; Cassis-Ghavami, Farah; Magaret, Amalia; Spies, Greg; Duerr, Ann; Celum, Connie; Sanchez, Jorge; Margolick, Joseph B.; Detels, Roger; McElrath, M. Juliana; Corey, Lawrence

    2011-01-01

    Background Adenoviruses are among the most promising vectors for the development of an HIV vaccine. The results of the phase IIB study of the adenovirus serotype 5-based Merck Trivalent HIV vaccine have raised the concern that serological immunity to Ad5 could be linked to HIV acquisition risk in high-risk individuals. We examined the association between adenovirus serostatus and the rate of incident HIV infection in populations at elevated risk of HIV acquisition. Methods We performed a nested case-control study of Ad5 serostatus among 299 HIV-infected and 590 matched HIV-uninfected persons participating in MACS and in HPTN 039, a study of HSV-2 suppression among adults in the U.S., South America and Africa. Appropriate HIV cases and controls were identified in each cohort, and Ad5 neutralizing antibody titers were compared in these two groups. Results In MACS and HPTN 039, the relative risks of incident HIV infection among Ad5 seropositive vs. Ad5 seronegative individuals were 1.1 (95% CI 0.8–1.5, p = 0.57) and 1.0 (95% CI 0.4 – 2.3, p = 0.99) respectively. HIV-1 acquisition rates did not vary significantly by Ad5 neutralizing antibody titer. Conclusions The presence of Ad5 neutralizing antibodies is not linked to the risk of HIV acquisition among populations at elevated risk of HIV infection. PMID:21150554

  18. Group C adenovirus DNA sequences in human lymphoid cells

    SciTech Connect

    Horvath, J.; Palkonyay, L.; Weber, J.

    1986-07-01

    Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 (Ad1), Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infectious in childhood result in the persistence of the viral genome in circulating lymphocytes.

  19. Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin.

    PubMed

    Agarwal, Payal; Gammon, Elizabeth A; Sajib, Abdul Mohin; Sandey, Maninder; Smith, Bruce F

    2017-01-01

    Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5's wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17-71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the

  20. Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin

    PubMed Central

    Gammon, Elizabeth A.; Sajib, Abdul Mohin; Sandey, Maninder; Smith, Bruce F.

    2017-01-01

    Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5’s wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17–71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17–71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17–71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address

  1. Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

    PubMed Central

    Lam, Eric

    2014-01-01

    ABSTRACT The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. IMPORTANCE This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions

  2. Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

    PubMed Central

    Kita, Ayami; Hino, Nobumasa; Higashi, Sakiko; Hirota, Kohji; Narumi, Ryohei; Adachi, Jun; Takafuji, Kazuaki; Ishimoto, Kenji; Okada, Yoshiaki; Sakamoto, Kensaku; Tomonaga, Takeshi; Takashima, Seiji; Mizuguchi, Hiroyuki; Doi, Takefumi

    2016-01-01

    The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest. PMID:27833131

  3. 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

    SciTech Connect

    Puntel, Mariana; Ghulam, Muhammad A.K.M.; Farrokhi, Catherine; VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley; Kroeger, Kurt M.; Salem, Alireza; Lacayo, Liliana; Pechnick, Robert N.; Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean; Palmer, Donna; Ng, Philip; 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

  4. The transduction of Coxsackie and Adenovirus Receptor-negative cells and protection against neutralizing antibodies by HPMA-co-oligolysine copolymer-coated adenovirus

    PubMed Central

    Wang, Chung-Huei K.; Chan, Leslie W.; Johnson, Russell N.; Chu, David S.H.; Shi, Julie; Schellinger, Joan G.; Lieber, Andre; Pun, Suzie H.

    2011-01-01

    Adenoviral (AdV) gene vectors offer efficient nucleic acid transfer into both dividing and non-dividing cells. However issues such as vector immunogenicity, toxicity and restricted transduction to receptor-expressing cells have prevented broad clinical translation of these constructs. To address this issue, engineered AdV have been prepared by both genetic and chemical manipulation. In this work, a polymer-coated Ad5 formulation is optimized by evaluating a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-co-oligolysine copolymers synthesized by living polymerization techniques. This synthesis approach was used to generate highly controlled and well-defined polymers with varying peptide length (K5, K10 and K15), polymer molecular weight, and degradability to coat the viral capsid. The optimal formulation was not affected by the presence of serum during transduction and significantly increased Ad5 transduction of several cell types that lack the Coxsackie and Adenovirus Receptor (CAR) by up to 6-fold compared to unmodified AdV. Polymer-coated Ad5 also retained high transduction capability in the presence of Ad5 neutralizing antibodies. The critical role of heparan sulfate proteoglycans (HSPGs) in mediating cell binding and internalization of polymer-coated AdV was also demonstrated by evaluating transduction in HSPG-defective recombinant CHO cells. The formulations developed here are attractive vectors for ex vivo gene transfer in applications such as cell therapy. In addition, this platform for adenoviral modification allows for facile introduction of alternative targeting ligands. PMID:21959008

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

    PubMed

    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.

  6. Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75

    PubMed Central

    Kumar, Ramesh; Sreenivasa, B. P.; Tamilselvan, R. P.

    2015-01-01

    Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV) capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm). Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5). Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01). Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by

  7. Localization of neutralization epitopes on adenovirus fiber knob from species C.

    PubMed

    Lang, Shuai; Wang, Lizheng; Wang, Zixuan; Zhu, Rui; Yan, Jingyi; Wang, Baoming; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Zhou, Yan; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-04-01

    Although potential neutralization epitopes on the fiber knob of adenovirus (AdV) serotype 2 (Ad2) and Ad5 have been revealed, few studies have been carried out to identify neutralization epitopes on the knob from a broader panel of AdV serotypes. In this study, based on sequence and structural analysis of knobs from Ad1, Ad2, Ad5 and Ad6 (all from species C), several trimeric chimeric knob proteins were expressed in Escherichia coli to identify the locations of neutralization epitopes on the knobs by analysing their reactivity with mouse and rabbit polyclonal sera raised against AdVs and human sera with natural AdV infection. The dominant neutralization epitopes were located mainly in the N-terminal part of knobs from Ad1, Ad2 and Ad5, but they seemed to be located in the C-terminal part of the Ad6 knob, with some individual differences in rabbit and human populations. Our study adds to our understanding of humoral immune responses to AdVs and will facilitate the construction of more desirable capsid-modified recombinant Ad5 vectors.

  8. Short-fiber protein of ad40 confers enteric tropism and protection against acidic gastrointestinal conditions.

    PubMed

    Rodríguez, Ester; Romero, Carolina; Río, Adolfo; Miralles, Marta; Raventós, Aida; Planells, Laura; Burgueño, Joan F; Hamada, Hirofumi; Perales, Jose Carlos; Bosch, Assumpció; Gassull, Miguel Angel; Fernández, Ester; Chillon, Miguel

    2013-08-01

    The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter β-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.

  9. Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than serotype 5: Implications for ovarian cancer therapy

    SciTech Connect

    Thoma, Clemens; Bachy, Veronique; Seaton, Patricia; Green, Nicola K.; Greaves, David R.; Klavinskis, Linda; Seymour, Leonard W.; Morrison, Joanne

    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.

  10. Determination of particle heterogeneity and stability of recombinant adenovirus by analytical ultracentrifugation in CsCl gradients.

    PubMed

    Yang, Xiaoyu; Agarwala, Shilpi; Ravindran, Sundari; Vellekamp, Gary

    2008-02-01

    Recombinant adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles. To determine particle heterogeneity we utilized analytical ultracentrifugation (AUC) in CsCl density gradients. Preparations of three different rAd vectors were assessed. AUC was able to resolve multiple density forms including two empty capsid types in various virus preparations. One unusual density form (form V), was noninfectious and lacked protein VI. AUC was able to quantify empty capsids and monitor their removal during process development. Their relative concentrations were reduced by either addition of an immobilized zinc affinity chromatography (IZAC) step or by extension of the infection time. The Adenovirus Reference Material (ARM), a wild-type Ad5, had 2.2% empty capsids and no other detectable minor particle forms. Finally, AUC was utilized to monitor the thermal instability of the three rAd vectors via the transformations of different density forms. The vector and empty capsids containing protein IX were more stable than those without IX. Together, these results exemplify AUC in CsCl density gradients as a valuable technique for evaluating product particle heterogeneity and stability.

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

    PubMed

    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.

  12. Traceless Bioresponsive Shielding of Adenovirus Hexon with HPMA Copolymers Maintains Transduction Capacity In Vitro and In Vivo

    PubMed Central

    Prill, Jan-Michael; Šubr, Vladimír; Pasquarelli, Noemi; Engler, Tatjana; Hoffmeister, Andrea; Kochanek, Stefan; Ulbrich, Karel; Kreppel, Florian

    2014-01-01

    future design of polymer-shielded Ad and provide a deeper insight into the interaction of shielded adenovirus vector particles with the host after systemic delivery. PMID:24475024

  13. Adenovirus mediated BIMS transfer induces growth supression and apoptosis in Raji lymphoma cells.

    PubMed

    Zhao, Ya Ning; Li, Qiang

    2014-09-01

    To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  14. 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.

    PubMed

    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.

  15. Use of dodecahedron "VLPs" as an alternative to the whole adenovirus.

    PubMed

    Fender, Pascal

    2014-01-01

    During human adenovirus type 3 (Ad3) infection, an excess of penton base and fiber proteins are produced. These form dodecahedral particles composed of 12 pentamers of penton base and 12 trimers of fiber protein. Beside this "natural" expression, the adenovirus dodecahedron can be expressed in the heterologous baculovirus system in two forms: a fiber-devoid dodecahedron made only of 12 penton bases (called base-dodecahedron: Bs-Dd) and the fiber-containing dodecahedron (called penton dodecahedron: Pt-Dd). These particles partly mimic the adenoviral cellular entry pathway but are devoid of genetic information making them an unusual tool for basic research or applications. We report here how these particles are expressed and purified, the labeling method for trafficking studies as well as their use in molecular interaction studies. The potential of these particles for biotechnological applications is under evaluation, making their study a "niche" along side traditional adenoviral vectors.

  16. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy.

    PubMed

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing

    2014-01-15

    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.

  17. Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression.

    PubMed Central

    Bruder, J T; Kovesdi, I

    1997-01-01

    Previous studies have shown that airway administration of adenovirus or adenovirus vectors results in a dose-dependent inflammatory response which limits the duration of transgene expression. We explored the possibility that adenovirus infection triggers signal transduction pathways that induce the synthesis of cytokines and thus contribute to the early inflammatory response. Since stimulation of the Raf/mitogen-activated protein kinase (MAPK) pathway activates transcription factors that control the expression of inflammatory cytokines, we examined the activation of this pathway following adenovirus infection. Adenovirus infection induced the rapid activation of Raf-1 and a transient increase in the tyrosine phosphorylation and activation of p42mapk at early times postinfection. Activation of the Raf/MAPK pathway by adenovirus is likely triggered by the infection process, since it occurred rapidly and with various mutant adenoviruses and adenovirus vectors. Moreover, interleukin-8 (IL-8) mRNA accumulation was evident at 20 min postinfection and was induced even in the presence of cycloheximide. Both MAPK activation and IL-8 production were inhibited by forskolin, a potent inhibitor of Raf-1. These results suggest that adenovirus-induced Raf/MAPK activation contributes to IL-8 production. Adenovirus-induced activation of the Raf/MAPK signaling pathway and IL-8 production may play critical roles in the inflammation observed following in vivo administration of adenovirus vectors for gene therapy. PMID:8985363

  18. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  19. Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.

    PubMed

    Schagen, F H; Rademaker, H J; Rabelink, M J; van Ormondt, H; Fallaux, F J; van der Eb, A J; Hoeben, R C

    2000-09-01

    In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

  20. Evaluation of polyhexamethylene biguanide (PHMB) as a disinfectant for adenovirus.

    PubMed

    Romanowski, Eric G; Yates, Kathleen A; O'Connor, Katherine E; Mah, Francis S; Shanks, Robert M Q; Kowalski, Regis P

    2013-04-01

    Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs. To determine whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs. In vitro laboratory study. The direct disinfecting activity of PHMB was determined in triplicate assays by incubating 9 human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with PHMB concentrations of 50 and 0 ppm (micrograms per milliliter) for 24 hours at room temperature to simulate swimming pool temperatures or 40oC to simulate hot tub temperatures. Plaque assays were performed to determine adenovirus titers after incubation. Titers were log10 converted and mean (SD) log10 reductions relative to controls were calculated. Virucidal (>99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested. RESULTS At room temperature, 50 ppm of PHMB produced mean reductions in titers less than 1 log10 for all adenovirus types tested. At 40°C, 50 ppm of PHMB produced mean reductions in titers less than 1 log10 for 2 adenovirus types and greater than 1 but less than 3 log10 for 7 of 9 adenovirus types. At a concentration of 50 ppm, PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Recreational water maintained and sanitized with PHMB can serve as a vector for the transmission of ocular adenovirus infections.

  1. Protection against Enterovirus 71 with Neutralizing Epitope Incorporation within Adenovirus Type 3 Hexon

    PubMed Central

    Tian, Xingui; Su, Xiaobo; Li, Xiao; Li, Haitao; Li, Ting; Zhou, Zhichao; Zhong, Tianhua; Zhou, Rong

    2012-01-01

    Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens. PMID:22848478

  2. Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

    PubMed Central

    Kryazhimskiy, Sergey; Grant, Rebecca; Calcedo, Roberto; Yuan, Xin; Keough, Martin; Sandhu, Arbans; Wang, Qiang; Medina-Jaszek, C. Angelica; Plotkin, Joshua B.; Wilson, James M.

    2009-01-01

    Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors. PMID:19578438

  3. Bioreducible polymer-conjugated oncolytic adenovirus for hepatoma-specific therapy via systemic administration.

    PubMed

    Kim, Pyung-Hwan; Kim, Jaesung; Kim, Tae-il; Nam, Hye Yeong; Yockman, James W; Kim, Minjung; Kim, Sung Wan; Yun, Chae-Ok

    2011-12-01

    Systemic administration of adenovirus (Ad) vectors is complicated by host immune responses and viral accumulation in the liver, resulting in a short circulatory virus half-life, low efficacy, and host side effects. Ad surface modification is thus required to enhance safety and therapeutic efficacy. An arginine-grafted bioreducible polymer (ABP) was chemically conjugated to the Ad surface, generating Ad-ΔE1/GFP-ABP. A hepatocellular carcinoma [HCC]-selective oncolytic Ad complex, YKL-1001-ABP, was also generated. Transduction efficiency of Ad-ΔE1/GFP-ABP was enhanced compared to naked Ad-ΔE1/GFP. YKL-1001-ABP elicited an enhanced and specific killing effect in liver cancer cells (Huh7 and HepG2) expressing α-fetoprotein (AFP). Compared with naked Ad, systemic administration of ABP-conjugated Ad resulted in reduced liver toxicity and interleukin (IL)-6 production in vitro and in vivo. Ad-ΔE1/GFP-ABP was more resistant to the neutralizing effects of human serum compared to naked Ad-ΔE1/GFP. ABP conjugation extended blood circulation time 45-fold and reduced anti-Ad Ab neutralization. Moreover, systemic administration of YKL-1001-ABP markedly suppressed growth of Huh7 hepatocellular carcinoma. These results demonstrate that chemical conjugation of ABP to the Ad surface improves safety and efficacy, indicating that ABP-conjugated Ad is a potentially useful cancer therapeutic agent to target cancer via systemic administration.

  4. Safety and immunogenicity of a chimpanzee adenovirus-vectored Ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study.

    PubMed

    De Santis, Olga; Audran, Régine; Pothin, Emilie; Warpelin-Decrausaz, Loane; Vallotton, Laure; Wuerzner, Grégoire; Cochet, Camille; Estoppey, Daniel; Steiner-Monard, Viviane; Lonchampt, Sophie; Thierry, Anne-Christine; Mayor, Carole; Bailer, Robert T; Mbaya, Olivier Tshiani; Zhou, Yan; Ploquin, Aurélie; Sullivan, Nancy J; Graham, Barney S; Roman, François; De Ryck, Iris; Ballou, W Ripley; Kieny, Marie Paule; Moorthy, Vasee; Spertini, François; Genton, Blaise

    2016-03-01

    The ongoing Ebola outbreak led to accelerated efforts to test vaccine candidates. On the basis of a request by WHO, we aimed to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire vaccine (ChAd3-EBO-Z). We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a trial at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Participants (aged 18-65 years) were randomly assigned (2:2:1), via two computer-generated randomisation lists for individuals potentially deployed in endemic areas and those not deployed, to receive a single intramuscular dose of high-dose vaccine (5 × 10(10) viral particles), low-dose vaccine (2·5 × 10(10) viral particles), or placebo. Deployed participants were allocated to only the vaccine groups. Group allocation was concealed from non-deployed participants, investigators, and outcome assessors. The safety evaluation was not masked for potentially deployed participants, who were therefore not included in the safety analysis for comparison between the vaccine doses and placebo, but were pooled with the non-deployed group to compare immunogenicity. The main objectives were safety and immunogenicity of ChAd3-EBO-Z. We did analysis by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02289027. Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive high-dose vaccine (n=49), low-dose vaccine (n=51), or placebo (n=20). Participants were followed up for 6 months. No vaccine-related serious adverse events were reported. We recorded local adverse events in 30 (75%) of 40 participants in the high-dose group, 33 (79%) of 42 participants in the low-dose group, and five (25%) of 20 participants in the placebo group. Fatigue or malaise was the most common systemic adverse event, reported in 25 (62

  5. Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system.

    PubMed

    Venkatraman, Giri; Behrens, Maik; Pyrski, Martina; Margolis, Frank L

    2005-09-01

    Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.

  6. Ad-shWnt2b Vector Therapy Demonstrates Antitumor Activity in Orthotopic Intrapleural Models as Monitored with the In Vitro Imaging System (IVIS).

    PubMed

    Kobayashi, Masashi; Huang, Cheng-Long; Sonobe, Makoto; Kikuchi, Ryutaro; Date, Hiroshi

    2016-11-01

    We investigated whether Wnt2B-inhibiting therapy has effective antitumor activity against Wnt2B-overexpressing cells in an orthotopic intrapleural model by monitoring with the in vitro imaging system (IVIS). Mice for the orthotopic intrapleural model were randomized into 3 groups (control, Ad-shWnt2B and Ad-scramble; 8 mice per group). The respective vector was injected into the intrapleural cavity of mice in the Ad-shWnt2B and Ad-scramble groups. After 6 weeks of vector treatment, all mice were monitored with the IVIS. Additionally, their body weight was measured until all mice died from the tumor or were sacrificed. A549-Luc-positive cells showed cytotoxicity following exposure to the Ad-shWnt2B vector. The percentage of viable cells was significantly lower in A549-Luc cells treated with Ad-shWnt2B than with Ad-scramble (p<0.01 versus control or Ad-scramble, respectively). Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    PubMed

    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.

  8. Adenovirus vector-mediated Gli1 siRNA induces growth inhibition and apoptosis in human pancreatic cancer with Smo-dependent or Smo-independent Hh pathway activation in vitro and in vivo.

    PubMed

    Guo, Jiefang; Gao, Jun; Li, Zhaoshen; Gong, Yanfang; Man, Xiaohua; Jin, Jing; Wu, Hongyu

    2013-10-10

    Activation of Hedgehog (Hh) signaling pathway is a core molecular mechanism in pancreatic carcinogenesis. However, the inhibition of upstream Hh signals does not inhibit the growth of a subset of pancreatic cancer (PC). This study was to examine the effect of siRNA targeting Gli1, the downstream component of Hh pathway, on PC cells and to provide some insight into the underlying mechanisms. A Gli1siRNA-expressing adenovirus (Ad-U6-Gli1siRNA) was constructed, and its effect on PC cells was investigated in vitro and in vivo. Gli1 was expressed in 83.3% (20/24) PC tissues, whereas no expression was found in normal pancreatic ductal epithelium. Gli1 was expressed in SW1990 and CFPAC cells in which Smo was completely absent, as well as in PaTu8988, Panc-1 and BxPC-3 cells in which Smo was concomitantly present. Ad-U6-Gli1siRNA induced cell growth inhibition, strong G0/G1 cell cycle arrest and apoptosis in all five human PC cell lines. Meanwhile, Ad-U6-Gli1siRNA significantly suppressed the expression of Gli1, Ptch1 and two target genes, Cyclin D2 and Bcl-2, in all five lines. Furthermore, two tumor xenograft nude mice models were established by subcutaneously injecting Smo-positive Panc-1 cells or Smo-negative SW1990 cells. The in vivo experimental results demonstrated that Ad-U6-Gli1siRNA inhibited the growth of both Panc1-derived and SW1990-derived tumors and induced cell apoptosis. Our study indicates that Gli1-targeting siRNA could induce growth inhibition and apoptosis in PC through knockdown of Gli1 and its target genes; and this method may represent a more effective therapeutic strategy for PC with Smo-dependent or Smo-independent Hh pathway activation.

  9. An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression.

    PubMed

    Hemminki, A; Belousova, N; Zinn, K R; Liu, B; Wang, M; Chaudhuri, T R; Rogers, B E; Buchsbaum, D J; Siegal, G P; Barnes, M N; Gomez-Navarro, J; Curiel, D T; Alvarez, R D

    2001-09-01

    The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to alpha(v)beta(3) and alpha(v)beta(5) integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials.

  10. Computational analysis of human adenovirus serotype 18

    PubMed Central

    Walsh, Michael P.; Seto, Jason; Tirado, Damaris; Chodosh, James; Schnurr, David; Seto, Donald; Jones, Morris S.

    2010-01-01

    The genome of the sole remaining unsequenced member of species A, human adenovirus type 18 (HAdV-A18), has been sequenced and analyzed. Members of species A are implicated as gastrointestinal pathogens and were shown to be tumorigenic in rodents. These whole genome and in silico proteome data are important as references for re-examining and integrating earlier work and observations based on lower resolution techniques, such as restriction enzyme digestion patterns, particularly for hypotheses based on pre-genomics data. Additionally, the genome of HAdV-A18 will also serve as reference for current studies examining the molecular evolution and origins of human and simian adenoviruses, particularly genome recombination studies. Applications of this virus as a potential vector for gene delivery protocols may be practical as data accumulates on this and other adenovirus genomes. PMID:20542532

  11. Helper-Dependent Adenovirus is Superior to First-Generation Adenovirus for Expressing Transgenes in Atherosclerosis-Prone Arteries

    PubMed Central

    Jiang, Bo; Qian, Kun; Du, Liang; Luttrell, Ian; Chitaley, Kanchan; Dichek, David A.

    2011-01-01

    Objective- Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation Ad that lacks all viral genes, is superior to first-generation Ad (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased. Methods and Results- Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. Relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both co-infusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested. Conclusions- HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries. PMID:21454808

  12. The Interaction between the Fiber Knob Domain and the Cellular Attachment Receptor Determines the Intracellular Trafficking Route of Adenoviruses

    PubMed Central

    Shayakhmetov, Dmitry M.; Li, Zong-Yi; Ternovoi, Vladimir; Gaggar, Anuj; Gharwan, Helen; Lieber, André

    2003-01-01

    Most of the presently used adenovirus (Ad) vectors are based on serotype 5. However, the application of these vectors is limited by the native tropism of Ad5. To address this problem, a series of fiber chimeric vectors were produced to take advantage of the different cellular receptors used by Ad of different subgroups. In this study we utilize an Ad5-based chimeric vector containing sequences encoding the Ad35 fiber knob domain instead of the Ad5 knob (Ad5/35L) to analyze factors responsible for selection of intracellular trafficking routes by Ads. By competition analysis with recombinant Ad5 and Ad35 knobs we showed that the Ad5/35L vector infected cells through a receptor different from the Ad5 receptor. Intracellular trafficking of Ad5 and Ad5/35L viruses was analyzed in HeLa cells by tracking fluorophore-conjugated Ad particles, by immunostaining for capsid hexon protein, by electron microscopy, and by Southern blotting for viral DNA. These studies showed that the interaction with the Ad35 receptor(s) predestines Ad5/35L vector to intracellular trafficking pathways different from those of Ad5. Ad5 efficiently escaped from the endosomes early after infection. In contrast, Ad5/35L remained longer in late endosomal/lysosomal compartments and used them to achieve localization to the nucleus. However, a significant portion of Ad5/35L particles appeared to be recycled back to the cell surface. This phenomenon resulted in significantly less efficient Ad5/35L-mediated gene transfer compared to that of Ad5. We also demonstrated that the selection of intracellular trafficking routes was determined by the fiber knob domain and did not depend on the length of the fiber shaft. This study contributes to a better understanding of the mechanisms that govern the infection of retargeted, capsid-modified vectors which have potential application for hematopoietic stem cell and tumor gene therapy. PMID:12610146

  13. Safety Profiles and Antitumor Efficacy of Oncolytic Adenovirus Coated with Bioreducible Polymer in the Treatment of a CAR Negative Tumor Model

    PubMed Central

    2015-01-01

    Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy. PMID:25400213

  14. Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

    PubMed

    Jung, Soo-Jung; Kasala, Dayananda; Choi, Joung-Woo; Lee, Soo-Hwan; Hwang, June Kyu; Kim, Sung Wan; Yun, Chae-Ok

    2015-01-12

    Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

  15. Cryo-EM structures of two bovine adenovirus type 3 intermediates

    SciTech Connect

    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

    2014-02-15

    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.

  16. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGES

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; ...

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  17. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

  18. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

    PubMed

    Xu, W; Neill, T; Yang, Y; Hu, Z; Cleveland, E; Wu, Y; Hutten, R; Xiao, X; Stock, S R; Shevrin, D; Kaul, K; Brendler, C; Iozzo, R V; Seth, P

    2015-03-01

    In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis.

  19. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    PubMed Central

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-01-01

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis. PMID:25503693

  20. Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

    DTIC Science & Technology

    2016-04-12

    disease has led to modify adenoviruses as vectors for vaccine development against other viral agents. Human adenovirus serotype 5 (HAd5) is the most...to contain the spread of viruses to the general population . Most licensed vaccines are made either by chemically inactivated whnle viruses or by...be catastrophic for viral biothreat agents that often cause the most lethal infections in humans . Therefore, new approaches are needed for the

  1. Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival

    PubMed Central

    Miller, Daniel L; Myers, Chad L; Rickards, Brenden; Coller, Hilary A; Flint, S Jane

    2007-01-01

    Background Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications. Results We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins. Conclusion These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors. PMID:17430596

  2. MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

    PubMed Central

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    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. PMID:23349911

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

    PubMed

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    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.

  4. Co-vaccination with adeno-associated virus vectors encoding human papillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF can elicit strong and prolonged neutralizing antibody.

    PubMed

    Liu, Dai-Wei; Chang, Junn-Liang; Tsao, Yeou-Ping; Huang, Chien-Wei; Kuo, Shu-Wen; Chen, Show-Li

    2005-01-01

    Non-infectious human papillomavirus-like particles (VLPs), encoded by the major capsid gene L1, have been shown to be effective as vaccines to prevent cervical cancer. We have developed the genetic immunization of the L1 gene to induce a neutralizing antibody. We constructed and generated a recombinant adeno-associated virus encoding human papillomavirus (HPV) 16 L1 protein that could form virus-like particles in transduced cells. Previous reports have demonstrated that the formation of VLP is necessary to induce high titers of neutralizing antibodies to protect an animal from viral challenge. Therefore, we carried out a single intramuscular (i.m.) injection with recombinant adeno-associated virus encoding HPV-16 L1 protein (rAAV-16L1) in BALB/c mice, which ultimately produced stronger and more prolonged neutralizing L1 antibodies, when compared to the DNA vaccine. Immunohistochemistry showed that the accumulation of antigen presenting cells, such as macrophages and dendritic cells, in rAAV-16L1 and L1 DNA-injected muscle fibers may be due to the L1 protein expression, but not to AAV infection. When compared to the L1 VLP vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV-16L1. Co-vaccinating with rAAV-16L1 and adenovirus encoding murine GM-CSF (rAAV-16L1/rAd-mGM-CSF) induced comparable higher levels of neutralizing L1 antibodies with those of VLP. This implies that a single i.m. co-injection with rAAV-16L1/rAd-mGM-CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections.

  5. A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

    PubMed

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won

    2014-05-07

    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  6. Use of an Automated Image Processing Program to Quantify Recombinant Adenovirus Particles

    NASA Astrophysics Data System (ADS)

    Obenauer-Kutner, Linda J.; Halperin, Rebecca; Ihnat, Peter M.; Tully, Christopher P.; Bordens, Ronald W.; Grace, Michael J.

    2005-02-01

    Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro® Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

  7. Emerging adenoviral vectors for stable correction of genetic disorders.

    PubMed

    Jager, Lorenz; Ehrhardt, Anja

    2007-08-01

    Recent drawbacks in treating patients with severe combined immunodeficiency disorders with retroviral vectors underline the importance of generating novel tools for stable transduction of mammalian cells. Substantial progress has been made over the recent years which may offer important steps towards stable and more importantly safer correction of genetic diseases. This article discusses recent advances for stable transduction of target cells based on adenoviral gene transfer. There is accumulating evidence that recombinant adenoviral vectors (AdVs) based on various human serotypes with a broad cellular tropism and adenoviruses (Ads) from different species will play an important role in future gene therapy applications. In combination with recombinant AdVs for somatic integration these gene transfer vectors offer high transduction efficiencies with potentially safer integration patterns. Other approaches for persistent transgene expression include excision of stable episomes from the adenoviral vector genome, but also long-term persistence of the complete adenoviral vector genome as an episomal DNA molecule was demonstrated and exemplified by the treatment of various genetic diseases in small and large animal models. This review displays advantages but also limitations of these Ad based vector systems. This is the perfect time to pursue such approaches because alternative strategies for stable transduction of mammalian cells undergoing many cell divisions are urgently needed. Looking into the future, we believe that a combination of different components from different viral vectors in concert with non-viral vector systems will be successful in designing significantly optimized transfer vehicles for a broad range of different genetic diseases.

  8. ChAd63-MVA–vectored Blood-stage Malaria Vaccines Targeting MSP1 and AMA1: Assessment of Efficacy Against Mosquito Bite Challenge in Humans

    PubMed Central

    Sheehy, Susanne H; Duncan, Christopher JA; Elias, Sean C; Choudhary, Prateek; Biswas, Sumi; Halstead, Fenella D; Collins, Katharine A; Edwards, Nick J; Douglas, Alexander D; Anagnostou, Nicholas A; Ewer, Katie J; Havelock, Tom; Mahungu, Tabitha; Bliss, Carly M; Miura, Kazutoyo; Poulton, Ian D; Lillie, Patrick J; Antrobus, Richard D; Berrie, Eleanor; Moyle, Sarah; Gantlett, Katherine; Colloca, Stefano; Cortese, Riccardo; Long, Carole A; Sinden, Robert E; Gilbert, Sarah C; Lawrie, Alison M; Doherty, Tom; Faust, Saul N; Nicosia, Alfredo; Hill, Adrian VS; Draper, Simon J

    2012-01-01

    The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also sh