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Sample records for replicating adenovirus vectors

  1. Adenovirus DNA Replication

    OpenAIRE

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new deve...

  2. Replication-Defective Vector Based on a Chimpanzee Adenovirus

    OpenAIRE

    Farina, Steven F.; Gao, Guang-Ping; Xiang, Z. Q.; Rux, John J.; Burnett, Roger M.; Alvira, Mauricio R.; Marsh, Jonathan; Ertl, Hildegund C.J.; Wilson, James M.

    2001-01-01

    An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including ...

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

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

    2016-08-15

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

  4. Inhibition of HIV-1 replication in alveolar macrophages by adenovirus gene transfer vectors.

    Science.gov (United States)

    Rice, Joshua; Connor, Ruth; Worgall, Stefan; Moore, John P; Leopold, Philip L; Kaner, Robert J; Crystal, Ronald G

    2002-08-01

    To assess the hypothesis that infection of alveolar macrophages (AM) with adenovirus (Ad) gene transfer vectors might prevent subsequent human immunodeficiency virus (HIV)-1 replication in AM, AM isolated from normal volunteers were infected with increasing doses of first generation (E1(-)) Ad vectors, followed 72 h later by infection with HIV-1(JRFL), an R5/M-tropic strain that preferentially uses the CCR5 coreceptor. As a measure of HIV-1 replication, p24 Ag was quantified by enzyme-linked imunosorbent assay in supernatants on Days 4 to 14 after HIV-1infection. Pretreatment of the AM with an Ad vector resulted in a dose- and time-dependent suppression of subsequent HIV-1 replication. The Ad vector inhibition of HIV-1 replication was independent of the transgene in the Ad vector expression cassette and E4 genes in the Ad backbone. Moreover, it did not appear to be secondary to a soluble factor released by the AM, nor was it overridden by the concomitant transfer of the CCR5 or CXCR4 receptors to the AM before HIV-1 infection. These observations have implications regarding pulmonary host responses associated with HIV-1 infection, as well as possibly uncovering new therapeutic strategies against HIV-1 infection.

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

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    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.

  6. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  7. Improvements in adenovirus-vector technology : aiming at replication specificity and vector integration

    NARCIS (Netherlands)

    Rademaker, Hendrik Jan

    2007-01-01

    Current generation adenoviral vectors (Ads) are not suitable for those gene therapy approaches that require long-term gene expression. This is due to their high immunogenicity and transient gene expression in fast dividing tissue. The development of gutless Ads, also known as helper-dependent Ads,

  8. Improvements in adenovirus-vector technology : aiming at replication specificity and vector integration

    NARCIS (Netherlands)

    Rademaker, Hendrik Jan

    2007-01-01

    Current generation adenoviral vectors (Ads) are not suitable for those gene therapy approaches that require long-term gene expression. This is due to their high immunogenicity and transient gene expression in fast dividing tissue. The development of gutless Ads, also known as helper-dependent Ads, i

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

    Directory of Open Access Journals (Sweden)

    Kahori Shimizu

    2014-01-01

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

  10. Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B19 replication.

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    Brandt, Marius R G; Kirste, Ariane G; Pozzuto, Tanja; Schubert, Steffen; Kandolf, Reinhard; Fechner, Henry; Bock, C-Thomas; Kurreck, Jens

    2013-09-01

    Human parvovirus B19 (B19V) has been considered to cause acute and chronic myocarditis, which is accompanied by endothelial dysfunction. Currently, no causative treatment option for B19V-infections is available. Since RNA interference (RNAi) has proven to be a highly potent antiviral approach, the aim of the current study was to develop an RNAi-based strategy to inhibit B19V replication. Three B19V-VP2-specific short hairpin RNAs (shRNAs) were designed and tested for their silencing activity in reporter assays and the expression cassette of the best one was introduced into an adenoviral shuttle vector (Ad5). B19V-permissive UT7/Epo-S1 cells were infected with B19V and the RNAi triggers were delivered by the adenoviral vector (Ad5shVP2) 24h thereafter. The shRNA targeting the B19V-VP2 gene significantly suppressed VP2 mRNA levels as determined by quantitative RT-PCR. Additionally, also the expression levels of the non-targeted non-structural B19V-NS1 mRNA were strongly reduced. Our results demonstrate that vector-mediated delivery of shRNA expression cassettes targeting the structural B19-VP2 gene is a suitable approach to inhibit B19V replication.

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

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    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.

  12. Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jim Silver

    Full Text Available Replication-competent adenovirus type 5 (Ad5 vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.

  13. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K.; Pearson, G D

    1985-01-01

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

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

    Science.gov (United States)

    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.

  15. Increasing the Efficacy of Oncolytic Adenovirus Vectors

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    William S. M. Wold

    2010-08-01

    Full Text Available Oncolytic adenovirus (Ad vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.

  16. Development of replication-deficient adenovirus malaria vaccines.

    Science.gov (United States)

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

    2017-03-01

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

  17. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  18. Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

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    Ono, Hidetaka A; Le, Long P; Davydova, Julia G; Gavrikova, Tatyana; Yamamoto, Masato

    2005-11-15

    To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

  19. Suppression of Adenovirus Replication by Cardiotonic Steroids.

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    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  20. A Replicating Adenovirus Capsid Display Recombinant Elicits Antibodies against Plasmodium falciparum Sporozoites in Aotus nancymaae Monkeys

    OpenAIRE

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

    2014-01-01

    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 antibodie...

  1. Replication-Uncoupled Histone Deposition during Adenovirus DNA Replication

    OpenAIRE

    Komatsu, Tetsuro; Nagata, Kyosuke

    2012-01-01

    In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of his...

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

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    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Replication of adenovirus DNA-protein complex with purified proteins.

    OpenAIRE

    Ikeda, J E; Enomoto, T.; Hurwitz, J

    1981-01-01

    A protein fraction isolated from the cytosol of adenovirus-infected HeLa cells, which contained DNA polymerase alpha, catalyzed adenoviral DNA replication in the presence of adenovirus DNA binding protein, eukaryotic DNA polymerase beta, ATP, all four dNTPs, and MgCl2. DNA replication started at either end of exogenously added adenoviral DNA and was totally dependent on the presence of terminal 55,000-dalton proteins on the DNA template. The replicaton of adenovirus DNA in the system was sens...

  4. Regulation of Human Adenovirus Replication by RNA Interference.

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    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  5. Amplified and Persistent Immune Responses Generated by Single-Cycle Replicating Adenovirus Vaccines

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    Crosby, Catherine M.; Nehete, Pramod; Sastry, K. Jagannadha

    2014-01-01

    ABSTRACT Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered “single-cycle” adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. IMPORTANCE This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase

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

    Directory of Open Access Journals (Sweden)

    Maria Abildgaard Steffensen

    Full Text Available Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii. To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

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

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    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.

  8. Molecular architecture of the preinitiation complex in adenovirus DNA replication

    OpenAIRE

    Mysiak, Monika Elzbieta

    2004-01-01

    After infection of a host cell, adenovirus (Ad) aims for generation of progeny viruses, and thus it rapidly replicates its genomic DNA. The replication process starts with the assembly of the preinitiation complex (PIC) on the origin DNA. The PIC consists of three viral proteins, DNA polymerase (pol), precursor terminal protein (pTP), DNA binding protein (DBP) and two transcription factors of the host cell, Nuclear Factor I (NFI) and Octamer binding protein (Oct-1). Both transcription factors...

  9. Exploiting features of adenovirus replication to support mammalian kinase production.

    Science.gov (United States)

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-11-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  11. Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells.

    Science.gov (United States)

    Guo, Ju-Tao; Zhou, Tianlun; Guo, Haitao; Block, Timothy M

    2007-12-01

    Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.

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

    Directory of Open Access Journals (Sweden)

    Sandy Ibanes

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

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

    Science.gov (United States)

    Ibanes, Sandy; Kremer, Eric J

    2013-01-01

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

  14. Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase.

    Science.gov (United States)

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen

    2012-10-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

  15. Inhibition of adenovirus replication in vitro by trifluridine.

    Science.gov (United States)

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  16. High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

    Institute of Scientific and Technical Information of China (English)

    CONG Tie-chuan; LU Zhe-ming; LI Yong; ZHENG Li; QIN Yong

    2007-01-01

    Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E.coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pacl and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture.After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

  17. Targeting species D adenoviruses replication to counteract the epidemic keratoconjunctivitis.

    Science.gov (United States)

    Nikitenko, Natalia A; Speiseder, Thomas; Groitl, Peter; Spirin, Pavel V; Prokofjeva, Maria M; Lebedev, Timofey D; Rubtsov, Petr M; Lam, Elena; Riecken, Kristoffer; Fehse, Boris; Dobner, Thomas; Prassolov, Vladimir S

    2015-06-01

    Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

  18. Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

    Science.gov (United States)

    Lemiale, Franck; Haddada, Hedi; Nabel, Gary J; Brough, Douglas E; King, C Richter; Gall, Jason G D

    2007-03-01

    Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.

  19. The Dual Nature of Nek9 in Adenovirus Replication

    OpenAIRE

    Jung, Richard; Radko, Sandi; Pelka, Peter

    2016-01-01

    To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to s...

  20. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

    NARCIS (Netherlands)

    van Beusechem, VW; van Rijswijk, ALCT; van Es, HHG; Haisma, HJ; Pinedo, HM; Gerritsen, WR

    2000-01-01

    Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfil

  1. Effect of CD4 gene expression on adenovirus replication.

    Science.gov (United States)

    Hotta, J; Shi, L; Ginsberg, H S

    1994-11-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.

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

    Science.gov (United States)

    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.

  3. Construction of conditionally replicative adenovirus vector mediated by dual specific promoters%双特异性启动子调控条件复制腺病毒载体的构建

    Institute of Scientific and Technical Information of China (English)

    李玮; 谭建

    2013-01-01

    Objective To construct and identify the conditionally replicative adenovirus vectors which may induce the human sodium iodide symporter (hNIS) expression in the early region 1A (E1A) gene of the human telomerase reverse transcriptase (hTERT) and the glial fibrillary acidic protein (GFAP)promoter regions.Methods Immunoblotting assay was employed to detect the variation in GFAP and telomerase protein expression prior to and following viral infection of the cerebral stellar glioblastoma cells (U87),neuroglioma cells (U251) and human embryonic lung fibroblasts (MRC-5) cells.The hTERT and GFAP promoters and the hNIS genes were amplified by polymerase chain reaction for synthesis of adenoviral E1A genes.The recombinant plasmids containing hTERT and GFAP gene promoters were adopted for transfection into MRC-5,U251 and U87,which entailed assessment of the hTERT and GFAP promoter activity via fluorescent analysis after 24 h.This was followed by ligation with the E1A and hNIS genes and subsequent cloning into the plasmid pDC311 for construction of the recombinant plasmid pDC311-Tp-E1A-Gp-NIS that was identified by double enzyme digestion (EcoR Ⅰ and Sal Ⅰ) and gene sequencing.This recombinant plasmid was co-transfected with adenoviral genomic plasmid pBHGlox△E1-3Cre into the human embryonic kidney 293 cells forming the conditionally replicative recombinant adenovirus Ad-Tp-E1A-Gp-NIS.This Ad-Tp-E1A-Gp-NIS was employed to transfect the 293,MRC-5,U87 and U251 cells,whose conditional replicability was measured by plaque forming assay.The recombinant virus Ad-Tp-E1A-Gp-NIS and Ad-CMV-EGFP controls were used to transfect the U251,U87 and MRC-5 cells for detection of the capacity of 125I uptake by using a γ-ray counter.Results The expression of the 120 000 telomerase and 49 000 GFAP protein could be found in U87 and U251 cells,but not in MRC-5 cells.Glioma target gene expression could be induced by the GFAP and hTERT promoters,the efficiency of which was (62.10±6.26)

  4. Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

    Science.gov (United States)

    Li, Shun; Ou, Mengting; Wang, Guixue; Tang, Liling

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future.

  5. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    Science.gov (United States)

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C

    2007-01-01

    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

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

    Science.gov (United States)

    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.

  7. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

    Science.gov (United States)

    Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

    2008-12-01

    With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

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

    Directory of Open Access Journals (Sweden)

    Eric J. Kremer

    2010-09-01

    Full Text Available 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.

  9. Role of preterminal protein processing in adenovirus replication.

    Science.gov (United States)

    Webster, A; Leith, I R; Nicholson, J; Hounsell, J; Hay, R T

    1997-09-01

    Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.

  10. Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

    Science.gov (United States)

    Guse, K; Dias, J D; Bauerschmitz, G J; Hakkarainen, T; Aavik, E; Ranki, T; Pisto, T; Särkioja, M; Desmond, R A; Kanerva, A; Hemminki, A

    2007-06-01

    Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Erkko Ylösmäki

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

  13. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    2015-11-01

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

  14. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    Science.gov (United States)

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  15. Low seroprevalent species D adenovirus vectors as influenza vaccines.

    Directory of Open Access Journals (Sweden)

    Eric A Weaver

    Full Text Available Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5 are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10(8 virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.

  16. Progress on adenovirus-vectored universal influenza vaccines.

    Science.gov (United States)

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

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

  17. Heat shock and heat shock protein 70i enhance the oncolytic effect of replicative adenovirus.

    Science.gov (United States)

    Haviv, Y S; Blackwell, J L; Li, H; Wang, M; Lei, X; Curiel, D T

    2001-12-01

    Replication-competent viruses are currently being evaluated for their cancer cell-killing properties. These vectors are designed to induce tumor regression after selective viral propagation within the tumor. However, replication-competent viruses have not resulted heretofore in complete tumor eradication in the clinical setting. Recently, heat shock has been reported to partially alleviate replication restriction on an avian adenovirus (Ad) in a human lung cancer cell line. Therefore, we hypothesized that heat shock and overexpression of heat shock protein (hsp) would support the oncolytic effect of a replication-competent human Ad. To this end, we tested the oncolytic and burst kinetics of a replication-competent Ad after exposure to heat shock or to inducible hsp 70 overexpression by a replication-deficient Ad (Adhsp 70i). Heat-shock resulted in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA. Overexpression of hsp 70i also enhanced Ad-mediated oncolysis but did not decrease intracellular Ad DNA levels. We conclude that heat shock and Adhsp 70i enhance the Ad cell-killing potential via distinct mechanisms. A potential therapeutic implication would be the use of local hyperthermia to augment oncolysis by increasing the burst of replication-competent Ad. The role of hsp in Ad-mediated oncolysis should be additionally explored.

  18. Effective gene-viral therapy for telomerase-positive cancers by selective replicative-competent adenovirus combining with endostatin gene

    Institute of Scientific and Technical Information of China (English)

    Zhang Q; Liu C; Jiang M; Fang G; Liu X; Wu M; Qian Q; Nie M; Sham J; Su C; Xue H; Chua D; Wang W; Cui Z; Liu Y

    2005-01-01

    Gene-viral therapy, which uses replication-selective transgene-expressing viruses to manage tumors, can exploit the virtues of gene therapy and virotherapy and overcome the limitations of conventional gene therapy. Using a human telomerase reverse transcriptase-targeted replicative adenovirus as an antiangiogenic gene transfer vector to target new angiogenesis and making use of its unrestrained proliferation are completely new concepts in tumor management. CNHK300-mE is a selective replication transgene-expressing adenovirus constructed to carry mouse endostatin gene therapeutically. Infection with CNHK300-mE was associated with selective replication of the adenovirus and production of mouse endostatin in telomerase-positive cancer cells. Endostatin secreted from a human gastric cell line, SGC-7901, infected with CNHK300-mE was significantly higher than that infected with nonreplicative adenovirus Ad-mE in vitro (800±94.7 ng/ml versus 132.9±9.9 ng/ml) and in vivo (610±42 ng/ml versus 126 +/- 13 ng/ml). Embryonic chorioallantoic membrane assay showed that the mouse endostatin secreted by CNHK300-mE inhibited angiogenesis efficiently and also induced distortion of pre-existing vasculature. CNHK300-mE exhibited a superior suppression of xenografts in nude mice compared with CNHK300 and Ad-mE. In summary, we provided a more efficient gene-viral therapy strategy by combining oncolysis with antiangiogenesis.

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

    Science.gov (United States)

    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.

  20. Going viral: a review of replication-selective oncolytic adenoviruses.

    Science.gov (United States)

    Larson, Christopher; Oronsky, Bryan; Scicinski, Jan; Fanger, Gary R; Stirn, Meaghan; Oronsky, Arnold; Reid, Tony R

    2015-08-21

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents.

  1. A novel tetracycline-controlled transactivator-transrepressor system enables external control of oncolytic adenovirus replication.

    Science.gov (United States)

    Fechner, H; Wang, X; Srour, M; Siemetzki, U; Seltmann, H; Sutter, A P; Scherübl, H; Zouboulis, C C; Schwaab, R; Hillen, W; Schultheiss, H-P; Poller, W

    2003-09-01

    The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication. We show here that a tumor-selective E1B-deleted RRCA can be tightly regulated by use of doxycycline (dox)-controlled adenoviral E1A gene expression, which in turn determines vector replication. RRCA replication is switched on by addition and switched off by withdrawal of dox. The system results in efficient tumor cell killing after induction by dox, whereas cells are unaffected by the uninduced system. It was also employed for efficient external control of transgene expression from cotransfected replication-deficient adenovectors. Furthermore, the use of a liver cell-specific human alpha1-antitrypsin (hAAT)-promoter driving a tetracycline-controlled transcriptional silencer allowed specific protection of cells with hAAT-promoter activity in the absence of dox in vitro and in vivo, delineating a new principle of 'tissue protective' gene therapy. The concept of external control of RRCAs may help to improve the safety of cancer gene therapy.

  2. The role of the adenovirus DNA binding protein in DNA replication and recombination

    NARCIS (Netherlands)

    Breukelen, B. van

    2003-01-01

    Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the

  3. Regulation of human adenovirus replication by RNA interference

    OpenAIRE

    Nikitenko, N. A.; SPEISEDER T.; Lam, E; Rubtsov, P. M.; TONAEVA KH. D.; S. A. Borzenok; Dobner, T; Prassolov, V.S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to...

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

    NARCIS (Netherlands)

    Gras, Jan Cornelis Emile

    2007-01-01

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

  5. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  6. Inhibitory effect of interferon-gamma on adenovirus replication and late transcription.

    Science.gov (United States)

    Mistchenko, A S; Diez, R A; Falcoff, R

    1989-06-15

    We have previously shown that human interferon-gamma inhibited adenovirus multiplication in vitro in a dose-dependent fashion. This action was previous to capsid proteins synthesis and did not involve virus adsorption nor penetration. In this report we have analysed viral mRNA levels at early (7 hr post infection (p.i.)) or late (20 hr p.i.) times, as well as DNA replication in Wish cells pretreated with interferon-gamma and infected with adenovirus 5. Controls included untreated cells as well as cells treated with interferon-alpha, to which adenovirus are reported to be resistant. Transcription of adenovirus regions E1, E4, L1 and L2 has been analysed by Northern blot. Adenovirus DNA replication was determined by DNA-DNA hybridization with total adenovirus 2 DNA. We have also searched for adenovirus E1A proteins by immunoblot with a specific monoclonal antibody. Although pretreatment of cells with either interferon-alpha or interferon-gamma resulted in reduced amounts of E1 and E4 mRNA in the early phase of infection (7 hr p.i.), the near complete inhibition of viral DNA and late transcription was only achieved by interferon-gamma. Immunoblot has shown the absence of the 48-kD E1A protein in cells pretreated with interferon-gamma. The lack of this regulatory adenovirus protein may be involved in the inhibitory mechanism of interferon-gamma on adenovirus.

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

    Directory of Open Access Journals (Sweden)

    Hiroyuki Mizuguchi

    2011-07-01

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

  8. Adenovirus-mediated shRNA interference against HSV-1 replication in vitro.

    Science.gov (United States)

    Song, Bo; Liu, Xinjing; Wang, Qingzhi; Zhang, Rui; Yang, Ting; Han, Zhiqiang; Xu, Yuming

    2016-12-01

    The UL29 and UL28 proteins encoded by herpes simplex virus type 1 (HSV-1) are critical for its replication and packaging, respectively. Research has demonstrated that synthesized siRNA molecules targeting the UL29 gene are able to suppress HSV-2 replication and the UL28-null HSV-1 gene cannot form infectious viruses in vitro. Silencing the UL28 and UL29 genes by RNAi might lead to the development of novel antiviral agents for the treatment of HSV-1 infections. Two kinds of short hairpin RNAs (shRNAs) targeting the UL29 and UL28 genes were chemically synthesized and then delivered into cells by a replication-defective human adenovirus type 5 (Adv5) vector. (-) shRNAs targeting none of the genome of HSV-1 were used as the control. Vero cells were inoculated with Ad-UL28shRNA or Ad-UL29shRNA at a multiplicity of infection (MOI) of 100 and challenged 24 h later with HSV-1 at an MOI of 0.01 to inhibit HSV-1 replication, as measured by the level of the corresponding RNA and proteins. In addition, the amount of progeny virus was assessed at daily intervals. The antiviral effects of Ad-shRNAs at ongoing HSV-1 infection were explored at 12 h after inoculation of the HSV-1. The results showed that the shRNAs delivered by Adv5 significantly suppressed HSV-1 replication in vitro, as determined by the levels of viral RNA transcription, viral protein synthesis, and viral production. The Ad-UL28shRNA and Ad-UL29shRNA suppressed the replication of HSV-1, respectively, compared with the control group (P HSV-1 infection (P HSV-1 infection.

  9. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  10. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

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

    Science.gov (United States)

    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...

  12. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  13. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    Science.gov (United States)

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

    Science.gov (United States)

    Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R Mark L; Toth, Karoly; Wold, William S M

    2014-12-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

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

    Directory of Open Access Journals (Sweden)

    Brian B Gowen

    Full Text Available 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.

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

    Science.gov (United States)

    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

  19. p53/E1b58kDa complex regulates adenovirus replication.

    Science.gov (United States)

    Ridgway, P J; Hall, A R; Myers, C J; Braithwaite, A W

    1997-10-27

    We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.

  20. Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

    Science.gov (United States)

    Murakami, Pete; Havenga, Menzo; Fawaz, Farah; Vogels, Ronald; Marzio, Giuseppe; Pungor, Erno; Files, Jim; Do, Linh; Goudsmit, Jaap; McCaman, Michael

    2004-01-01

    The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 × 1012 vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad. PMID:15163713

  1. SD大鼠白细胞介素10复制缺陷型重组腺病毒的构建及鉴定术%Construction and identification of recombinant replication- defective adenovirus vector containing interleukin-10 in a rat

    Institute of Scientific and Technical Information of China (English)

    李爽; 宋仁刚; 唐冰; 朱斌; 朱旭红; 朱家源; 毕良宽

    2009-01-01

    背景:重组腺病毒质粒的构建是重组腺病毒制备过程中的中枢环节,传统的细胞内同源重组法步骤多、成功率低、实验周期长.目的:构建含有大鼠白细胞介素10(rlL-10)基因的重组复制缺陷型腺病毒Ad.rlL-10,为真核表达及其动物模型的研究提供实验基础.设计、时间及地点:开放性实验,于2005-07/2006-04在中山大学附属第一医院完成.材料:SD大鼠1只.AdEasy系统由美国John Hopkins肿瘤研究中心惠赠.ThermoscriptTMRT kit 和Trizol 为Invitrogen产品;HEK-293保存于中山大学动物实验中心细胞库;克隆大鼠白细胞介素10基因引物合成及测序由上海博亚生物技术有限公司完成.方法:采用反转录聚合酶链反应的方法从健康SD大鼠脾脏新鲜组织中提取的总RNA中克隆rIL-10基因,采用AdEasy-1腺病毒载体系统经E.CoilBJ5183细菌内同源重组及HEK.293包装而获得重组腺病毒Ad.rlL-10.主要观察指标:用Western blot和RT-PCR的方法鉴定Ad.rlL-10并进行滴度测定.结果:自健康SD大鼠脾脏组织中克隆rIL-10基因,构建出重组腺病毒Ad.rlL-10.经PCR扩增、酶切鉴定、DNA 测序最终确定插入序列为rlL-1 0,Western blot和RT-PCR均检测出细胞内rlL-10基因及其蛋白表达.再通过扩增、纯化后获得滴度为1.0x1014pfu/mL的重组腺病毒.结论:采用AdEasy-1系统,经细HEK-293细胞包装、扩增,纯化后获得足够数量和质量的病毒,方法简便易行,结果稳定可靠.%BACKGROUND: Construction of recombinant adenovirus plasmid plays a central role in preparation of recombinant adenovirus.However, conventionally intracellular homologous recombination method is limited by complex procedures, low successful ratio,and long experimental cycle.OBJECTIVE: To construct the recombinant replication-defective adenovirus vector containing interleukin-10 (Ad.dL-10) in a SD rat, and to provide experimental evidences for eukaryotic expression and animat model studying

  2. Transduction and apoptosis induction in the rat prostate, using adenovirus vectors.

    Science.gov (United States)

    Kirkman, W; Chen, P; Schroeder, R; Feneley, M R; Rodriguez, R; Wickham, T J; King, C R; Bruder, J T

    2001-08-10

    Proapoptotic adenovirus vectors offer great promise for the treatment of cancer and nonmalignant conditions. Benign prostate hyperplasia (BPH) is a common nonmalignant enlargement of the prostate that involves epithelial, stromal, and smooth muscle components of the gland. We tested the hypothesis that an adenovirus vector expressing Fas ligand can be used to induce apoptosis in the prostate. We analyzed the efficiency of transduction and apoptosis induction in primary cultures of human prostate cells after adenovirus-mediated gene transfer. Efficient transduction was observed in primary prostate epithelial cells. Stromal and smooth muscle cells were more difficult to transduce, as no coxsackie-adenovirus receptor (CAR) expression was detectable on these cells. However, transduction was achieved in these cells when the multiplicity of infection was increased to 100 focal-forming units per cell, or when the vectors were delivered as calcium phosphate precipitates. Infection of all three primary prostate cell types with an adenovirus vector that expresses Fas ligand (AdFasL/G) resulted in rapid apoptosis. Direct injection of the rat prostate with an adenovirus vector carrying luciferase resulted in substantial luciferase expression. TUNEL analysis demonstrated that AdFasL/G administration induced low-level apoptosis in prostatic epithelial cells throughout the gland. As a first step toward enhancing the efficiency of prostate transduction in vivo, we tested an adenovirus vector that was engineered to have an expanded tropism. This vector, AdZ.F2K(pK7), was 10- to 500-fold more efficient than unmodified vectors in transducing prostate epithelial, smooth muscle, and stromal cells in culture. Moreover, AdZ.F2K(pK7) was more efficient than an unmodified vector at transducing the rat prostate in vivo, although the effect was dose dependent.

  3. Estramustine phosphate reversibly inhibits an early stage during adenovirus replication.

    Science.gov (United States)

    Everitt, E; Ekstrand, H; Boberg, B; Hartley-Asp, B

    1990-01-01

    Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

  4. shRNA-armed conditionally replicative adenoviruses: a promising approach for cancer therapy

    Science.gov (United States)

    Zhang, Jie; Ding, Meng; Xu, Kai; Mao, Lijun; Zheng, Junian

    2016-01-01

    The small-interfering RNAs (siRNAs) have been employed to knockdown the expression of cancer-associated genes and shown some promise in cancer therapy. However, synthetic siRNA duplexes or plasmid mediated delivery of siRNAs have several problems, such as short half-life, low transfection efficiency and cytotoxicity associated with transfection. Conditionally replicating adenovirus (CRAds) as the delivery vector for short hairpin RNAs (shRNAs) could overcome these limitations and have shown augmented anti-tumor effects in experimental studies and preclinical trials. In this review, we summarize recent progress in the development of CRAds-shRNA for cancer treatment. Combination of CRAds-shRNA with chemotherapeutics, radiation, dendritic cells, monoclonal antibodies and small-molecule inhibitors will be necessary to eradicate cancer cells and cancer stem cells and achieve superior outcomes. The use of CRAd platform for efficient delivery of shRNAs and foreign genes will open a new avenue for cancer therapy. PMID:26980708

  5. 携带TRAIL基因的条件复制型腺病毒载体的构建及其辐射诱导表达%Construction of conditionally replicative adenovirus vector carrying TRAIL gene and its mRNA and protein expressions induced by ionizing radiation

    Institute of Scientific and Technical Information of China (English)

    王宏芳; 吴嘉慧; 刘纯岩; 刘威武; 孙延红; 龚守良; 王志成; 刘扬

    2014-01-01

    Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set up:control, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.%目的:构建携带早期生长反应基因-1(Egr-1)启动子和肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因的条件复制型腺病毒载体 pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K

  6. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

    Science.gov (United States)

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

    Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

  7. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  8. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai

    2006-01-01

    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  9. Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma

    Institute of Scientific and Technical Information of China (English)

    Cui Hai; Yong-Min Jin; Wen-Biao Jin; Zhe-Zhu Han; Mei-Nv Cui; Xue-Zhe Piao; Xiong-Hu Shen; Song-Nan Zhang; Hong-Hua Sun

    2012-01-01

    Although gene therapy was regarded as a promising approach for glioma treatment,its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems.Mesenchymal stem cells (MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy.Therefore,in this study,we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus.We firstly compared the infectivity of type 3,type 5,and type 35 fiber-modified adenoviruses in MSCs.We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo.Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus.MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups.In conclusion,MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma,making it a potential therapeutic strategy for treating malignant glioma.

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

    NARCIS (Netherlands)

    Kim, Julius W.; Glasgow, Joel N.; 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

  11. Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

    Science.gov (United States)

    Doucas, V; Ishov, A M; Romo, A; Juguilon, H; Weitzman, M D; Evans, R M; Maul, G G

    1996-01-15

    Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.

  12. Valganciclovir Inhibits Human Adenovirus Replication and Pathology in Permissive Immunosuppressed Female and Male Syrian Hamsters

    Directory of Open Access Journals (Sweden)

    Karoly Toth

    2015-03-01

    Full Text Available Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5 infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

  13. Valganciclovir inhibits human adenovirus replication and pathology in permissive immunosuppressed female and male Syrian hamsters.

    Science.gov (United States)

    Toth, Karoly; Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Sagartz, John E; Buller, Robert Mark L; Wold, William S M

    2015-03-23

    Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

  14. Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Huajun Jin

    Full Text Available Highly selective therapy for hepatocellular carcinoma (HCC remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4% and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011(let7T, by introducing eight copies of let-7 target sites (let7T into the 3' untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels of the SG7011(let7T was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011(let7T was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68 expressing high level of let-7 (>300 folds, whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5 with low level of let-7. Consequently, the cytotoxicity of SG7011(let7T to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011(let7Tin vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011(let7T may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.

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

    Science.gov (United States)

    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.

  16. An Adenovirus-Vectored Influenza Vaccine Induces Durable Cross-Protective Hemagglutinin Stalk Antibody Responses in Mice

    Directory of Open Access Journals (Sweden)

    Eun Hye Kim

    2017-08-01

    Full Text Available Currently licensed vaccines against the influenza A virus (IAV need to be updated annually to match the constantly evolving antigenicity of the influenza virus glycoproteins, hemagglutinin (HA, and neuramidiase (NA. Attempts to develop universal vaccines that provide broad protection have resulted in some success. Herein, we have shown that a replication-deficient adenovirus expressing H5/M2e induced significant humoral immunity against the conserved HA stalk. Compared to the humoral responses induced by an inactivated influenza vaccine, the humoral responses induced by the adenovirus-vectored vaccine against the conserved stalk domain mediated cross-protection against heterosubtypic influenza viruses. Importantly, virus inactivation by formaldehyde significantly reduced the binding of monoclonal antibodies (mAbs to the conserved nucleoprotein (NP, M2e, and HA stalk. These results suggest that inactivation by formaldehyde significantly alters the antigenicity of the HA stalk, and suggest that the conformation of the intact HA stalk provided by vector-based vaccines is important for induction of HA stalk-binding Abs. Our study provides insight into the mechanism by which a vector-based vaccine induces broad protection by stimulation of cross-protective Abs targeting conserved domains of viral proteins. The findings support further strategies to develop a vectored vaccine as a universal influenza vaccine for the control of influenza epidemics and unpredicted pandemics.

  17. An Adenovirus-Vectored Influenza Vaccine Induces Durable Cross-Protective Hemagglutinin Stalk Antibody Responses in Mice.

    Science.gov (United States)

    Kim, Eun Hye; Han, Gye-Yeong; Nguyen, Huan

    2017-08-21

    Currently licensed vaccines against the influenza A virus (IAV) need to be updated annually to match the constantly evolving antigenicity of the influenza virus glycoproteins, hemagglutinin (HA), and neuramidiase (NA). Attempts to develop universal vaccines that provide broad protection have resulted in some success. Herein, we have shown that a replication-deficient adenovirus expressing H5/M2e induced significant humoral immunity against the conserved HA stalk. Compared to the humoral responses induced by an inactivated influenza vaccine, the humoral responses induced by the adenovirus-vectored vaccine against the conserved stalk domain mediated cross-protection against heterosubtypic influenza viruses. Importantly, virus inactivation by formaldehyde significantly reduced the binding of monoclonal antibodies (mAbs) to the conserved nucleoprotein (NP), M2e, and HA stalk. These results suggest that inactivation by formaldehyde significantly alters the antigenicity of the HA stalk, and suggest that the conformation of the intact HA stalk provided by vector-based vaccines is important for induction of HA stalk-binding Abs. Our study provides insight into the mechanism by which a vector-based vaccine induces broad protection by stimulation of cross-protective Abs targeting conserved domains of viral proteins. The findings support further strategies to develop a vectored vaccine as a universal influenza vaccine for the control of influenza epidemics and unpredicted pandemics.

  18. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    Directory of Open Access Journals (Sweden)

    Natascha Krömmelbein

    2016-02-01

    Full Text Available The human cytomegalovirus (HCMV replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

  19. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

    Science.gov (United States)

    Pombo, A; Ferreira, J; Bridge, E; Carmo-Fonseca, M

    1994-11-01

    We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

  20. RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments.

    Science.gov (United States)

    Rauschhuber, Christina; Mueck-Haeusl, Martin; Zhang, Wenli; Nettelbeck, Dirk M; Ehrhardt, Anja

    2013-01-01

    RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

  1. Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

    Science.gov (United States)

    Li, H; Haviv, Y S; Derdeyn, C A; Lam, J; Coolidge, C; Hunter, E; Curiel, D T; Blackwell, J L

    2001-12-10

    Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

  2. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    Science.gov (United States)

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    Science.gov (United States)

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  5. CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

    Directory of Open Access Journals (Sweden)

    Young Lawrence S

    2010-03-01

    Full Text Available Abstract Background CD40 and its ligand (CD40L play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours.

  6. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Directory of Open Access Journals (Sweden)

    Ling Zhang

    Full Text Available Recombinant interferon-γ (IFNγ production in cultured lentivirus (LV was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5. The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041, and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032, which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  7. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Science.gov (United States)

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  8. New Conditionally Replicating Adenovirus Vectors for Breast Cancer Therapy

    Science.gov (United States)

    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

  9. Differential Innate Immune Stimulation Elicited by Adenovirus and Poxvirus Vaccine Vectors

    OpenAIRE

    Teigler, Jeffrey Edward

    2014-01-01

    Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited b...

  10. Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus ▿

    OpenAIRE

    2009-01-01

    Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infe...

  11. Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

    Science.gov (United States)

    Bachmann, Mandy; Breitwieser, Theresa; Lipps, Christoph; Wirth, Dagmar; Jordan, Ingo; Reichl, Udo; Frensing, Timo

    2016-02-01

    Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

  12. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    Science.gov (United States)

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  13. Adenovirus replication as an in vitro probe for drug sensitivity in human tumors.

    Science.gov (United States)

    Parsons, P G; Maynard, K R; Little, J H; McLeod, G R

    1986-04-01

    The feasibility of using adenovirus 5 as an in vitro probe for chemosensitivity in short-term cultures of human tumors was evaluated using human melanoma cell lines and primary cultures of melanoma biopsies. A convenient immunoperoxidase method was developed for quantitating viral replication 2 days after infection. Two different approaches were explored: the host cell reactivation assay (HCR) using drug-treated virus; and the viral capacity assay using drug-treated cells. The HCR assay detected sensitivity to 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) in Mer- (methyl excision repair deficient) cell lines as decreased ability of the cells to replicate MTIC-treated virus. This test should be applicable to DNA-damaging agents and repair-deficient tumors. Adenovirus replicated readily in nonproliferating primary cultures of melanoma biopsies; application of the HCR assays to this material identified one Mer- sample of 11 tested. Herpes viruses were not suitable for use in HCR because herpes simplex virus type 1 failed to distinguish Mer- from Mer+ melanoma cells; and nonproductive infection of MTIC-sensitive lymphoid cells with Epstein-Barr virus yielded an MTIC-resistant cell line. The second assay (viral capacity) involved determination of the inhibition of replication of untreated virus in treated cells. This approach correctly predicted sensitivity to hydroxyurea and deoxyadenosine in melanoma cell lines when compared with clonogenic survival assay. Viral capacity was also inhibited by cytosine arabinoside, fluorouracil, vincristine, adriamycin, 6-mercaptopurine and ionising radiation, and may therefore be useful for detecting sensitivity to a wide range of antitumor agents.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Julius W Kim

    Full Text Available 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.

  16. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    OpenAIRE

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping

    2013-01-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...

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

    Science.gov (United States)

    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.

  18. THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Jiang Lei; Xia Yongjing; Li Hongxia; Hu Yajun; Hu Shixue; Xu Hongji

    1998-01-01

    Objective:To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene,on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

  19. CAR chasing: canine adenovirus vectors-all bite and no bark?

    Science.gov (United States)

    Kremer, Eric J

    2004-02-01

    This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (DeltaE1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues.

  20. Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

    Institute of Scientific and Technical Information of China (English)

    哈小琴; 苑宾; 李元敏; 劳妙芬; 吴祖泽

    2003-01-01

    A complementary DNA (cDNA) encoding human hepatocyte growth factor wasintroduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities tohuman hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF,the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that ofthe normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereasno therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2006-06-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  5. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine.

    Science.gov (United States)

    Toro, Haroldo; Suarez, David L; Tang, De-chu C; van Ginkel, Frederik W; Breedlovea, Cassandra

    2011-03-01

    We evaluated protection conferred by mucosal vaccination with replication-competent adenovirus-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdTW68.H5ck). Commercial, layer-type chicken groups were either singly vaccinated ocularly at 5 days of age, singly vaccinated via spray at 5 days of age, or ocularly primed at 5 days and ocularly boosted at 15 days of age. Only chickens primed and boosted via the ocular route developed AI systemic antibodies with maximum hemagglutination inhibition mean titers of 3.9 log2 at 32 days of age. In contrast, single vaccination via the ocular or spray routes maintained an antibody status similar to unvaccinated controls. All chickens (16/16) subjected to ocular priming and boosting with AdTW68.H5ck survived challenge with highly pathogenic AI virus A/chicken/Queretaro/14588-19/95 (H5N2). Single ocular vaccination resulted in 63% (10/16) of birds surviving the challenge followed by a 44% (7/16) survival of single-sprayed vaccinated birds. Birds vaccinated twice via the ocular route also showed significantly lower (P < 0.05) AI virus RNA concentrations in oropharyngeal swabs compared to unvaccinated-challenged controls.

  6. Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

    Directory of Open Access Journals (Sweden)

    Victor M. Bii

    2016-10-01

    Full Text Available Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

  7. Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative

    Science.gov (United States)

    Sanchez-Cespedes, Javier; Moyer, Crystal L.; Whitby, Landon R.; Boger, Dale L.; Nemerow, Glen R.

    2014-01-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections. PMID:24907427

  8. The long repeat region is dispensable for fowl adenovirus replication in vitro.

    Science.gov (United States)

    Ojkic, D; Nagy, E

    2001-05-10

    Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome. The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown. An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses. The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2. Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro. The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR.

  9. Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative.

    Science.gov (United States)

    Sanchez-Cespedes, Javier; Moyer, Crystal L; Whitby, Landon R; Boger, Dale L; Nemerow, Glen R

    2014-08-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.

  10. Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines.

    Science.gov (United States)

    Musk, P; Stowers, A; Parsons, P G

    1990-02-15

    Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.

  11. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

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

    Science.gov (United States)

    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

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

    OpenAIRE

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Joseph T Bruder; 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 genetica...

  14. Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

    Institute of Scientific and Technical Information of China (English)

    Xiao HU; Lei Lou; Jun Yuan; Xing Wan; Jianyi Wang; Xinyue Qin

    2010-01-01

    Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation.Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to clarify the undedying mechanisms,in the present study,an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells.Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection,followed by 100 multiplicity of infection.Results showed overexpression of estrogen receptor a mRNA and protein in the infected nerve cells.Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.

  15. Conditionally replicating adenovirus prevents pluripotent stem cell–derived teratoma by specifically eliminating undifferentiated cells

    Directory of Open Access Journals (Sweden)

    Kaoru Mitsui

    2015-01-01

    Full Text Available Incomplete abolition of tumorigenicity creates potential safety concerns in clinical trials of regenerative medicine based on human pluripotent stem cells (hPSCs. Here, we demonstrate that conditionally replicating adenoviruses that specifically target cancers using multiple factors (m-CRAs, originally developed as anticancer drugs, may also be useful as novel antitumorigenic agents in hPSC-based therapy. The survivin promoter was more active in undifferentiated hPSCs than the telomerase reverse transcriptase (TERT promoter, whereas both promoters were minimally active in differentiated normal cells. Accordingly, survivin-responsive m-CRA (Surv.m-CRA killed undifferentiated hPSCs more efficiently than TERT-responsive m-CRAs (Tert.m-CRA; both m-CRAs exhibited efficient viral replication and cytotoxicity in undifferentiated hPSCs, but not in cocultured differentiated normal cells. Pre-infection of hPSCs with Surv.m-CRA or Tert.m-CRA abolished in vivo teratoma formation in a dose-dependent manner following hPSC implantation into mice. Thus, m-CRAs, and in particular Surv.m-CRAs, represent novel antitumorigenic agents that could facilitate safe clinical applications of hPSC-based regenerative medicine.

  16. Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

    Science.gov (United States)

    Brusca, J S; Chinnadurai, G

    1981-01-01

    We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection. Images PMID:7277578

  17. Immunosuppression enhances oncolytic adenovirus replication and antitumor efficacy in the Syrian hamster model.

    Science.gov (United States)

    Thomas, Maria A; Spencer, Jacqueline F; Toth, Karoly; Sagartz, John E; Phillips, Nancy J; Wold, William S M

    2008-10-01

    We recently described an immunocompetent Syrian hamster model for oncolytic adenoviruses (Ads) that permits virus replication in tumor cells as well as some normal tissues. This model allows exploration of interactions between the virus, tumor, normal organs, and host immune system that could not be examined in the immunodeficient or nonpermissive animal models previously used in the oncolytic Ad field. Here we asked whether the immune response to oncolytic Ad enhances or limits antitumor efficacy. We first determined that cyclophosphamide (CP) is a potent immunosuppressive agent in the Syrian hamster and that CP alone had no effect on tumor growth. Importantly, we found that the antitumor efficacy of oncolytic Ads was significantly enhanced in immunosuppressed animals. In animals that received virus therapy plus immunosuppression, significant differences were observed in tumor histology, and in many cases little viable tumor remained. Notably, we also determined that immunosuppression allowed intratumoral virus levels to remain elevated for prolonged periods. Although favorable tumor responses can be achieved in immunocompetent animals, the rate of virus clearance from the tumor may lead to varied antitumor efficacy. Immunosuppression, therefore, allows sustained Ad replication and oncolysis, which leads to substantially improved suppression of tumor growth.

  18. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  19. Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

    Directory of Open Access Journals (Sweden)

    Takeshi Nishimoto

    Full Text Available The targeting of gene transfer at the cell-entry level 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 the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

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

    Directory of Open Access Journals (Sweden)

    Bhatia S

    2016-11-01

    Full Text Available Shilpa Bhatia,1 Samia M O’Bryan,1 Angel A Rivera,2 David T Curiel,3 J Michael Mathis1 1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 2Departments of Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL, 3Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO, USA Abstract: 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. Keywords: adapter, adenovirus serotype 5, cancer, hCAR, human coxsackievirus and adenovirus receptor, chemokine receptor, CXCL12, CXCR4, gene therapy, retargeting, viral vector

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

    Directory of Open Access Journals (Sweden)

    Briana Jill Williams

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

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

    Science.gov (United States)

    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.

  3. Construction of a recombinant adenovirus Vector of human papillomavirus type 16 L1_E7c

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 L1 proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7c. Methods: Human papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559- 7152) and E7c (682- 855) were amplified using PCR. The L1 and E7c fragments were inserted into pGEM-T easy vectors by T- A strategy, named pTAL1 and pTAE7c. pTAL1 was cut with Hind III and BglII, the pTAE7c with BamHI and ClaI. The L1 DNA fragment, E7c and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7c and pBSL1-E7c was digested with Hind III and Xhol. The L1-E7c fragment was inserted into adenovirus shuttle plasmids pCAl4, named pCAl4L1-E7c. DNA sequence results indicated that The L1-E7c DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7c DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7c, pBSL1-E7c and pCA14L1-E7c were constructed correctly. The pCAl4L1-E7c can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALl-E7c for the further research.

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

    Directory of Open Access Journals (Sweden)

    Stefania Piersanti

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

  5. Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters.

    Science.gov (United States)

    Shoji, Masaki; Yoshizaki, Shinji; Mizuguchi, Hiroyuki; Okuda, Kenji; Shimada, Masaru

    2012-01-01

    Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.

  6. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    Science.gov (United States)

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  7. Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

    Institute of Scientific and Technical Information of China (English)

    朱瑾; 吴军; 陈希炜; 易绍萱; 罗高兴; 贺伟峰

    2003-01-01

    Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

  8. Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

    Science.gov (United States)

    Gonzalez, R; Vereecque, R; Wickham, T J; Vanrumbeke, M; Kovesdi, I; Bauters, F; Fenaux, P; Quesnel, B

    1999-03-01

    Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

  9. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    Science.gov (United States)

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

  10. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    Science.gov (United States)

    Wickham, T J; Tzeng, E; Shears, L L; Roelvink, P W; Li, Y; Lee, G M; Brough, D E; Lizonova, A; Kovesdi, I

    1997-11-01

    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.

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

    Science.gov (United States)

    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. Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

    Science.gov (United States)

    Jerkofsky, M; Rapp, F

    1975-02-01

    Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2002-11-01

    The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

  15. De Novo Synthesis of Marburg Virus Antigens from Adenovirus Vectors Induce Potent Humoral and Cellular Immune Responses

    Science.gov (United States)

    2005-12-09

    cells and purified by ultra-centrifugation in esium chloride gradients. Briefly, adenoviral lysates from hirty 150-mm plates were banded twice on CsCl...differences of individuals with cystic fibrosis con- sequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther

  16. An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

    Science.gov (United States)

    Cannio, Raffaele; Contursi, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

    1998-01-01

    A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. PMID:9620978

  17. Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses effectively suppress growth of malignant pleural mesothelioma.

    Science.gov (United States)

    Harada, Akiko; Uchino, Junji; Harada, Taishi; Nakagaki, Noriaki; Hisasue, Junko; Fujita, Masaki; Takayama, Koichi

    2017-01-01

    Malignant mesothelioma (MM) incidence is increasing drastically worldwide as an occupational disease resulting from asbestos exposure. However, no curative treatment for MM of advanced stage is available. Thus, new therapeutic approaches for MM are required. Because malignant pleural mesothelioma (MPM) cells spread along the pleural surface in most patients, MPM can be targeted using intrapleural therapeutic approaches. In this study, we investigated the effectiveness of the intrapleural instillation of a replication-competent adenovirus as an oncolytic agent against MPM. We constructed a vascular endothelial growth factor promoter-based conditionally replicative adenovirus (VEGF-CRAd) that replicates exclusively in VEGF-expressing cells. All of the MM cell lines that we tested expressed VEGF mRNA, and VEGF-CRAd selectively replicated in these MM cells and exerted a direct concentration-dependent oncolytic effect in vitro. Furthermore, our in vivo studies showed that pre-infection of MM cells with VEGF-CRAd potently suppressed MPM tumor formation in nude mice, and that intrapleural instillation of VEGF-CRAd prolonged the survival time of tumor-bearing mice. Our results indicate that VEGF-CRAd exerts an oncolytic effect on MM cells and that intrapleural instillation of VEGF-CRAd is safe and might represent a promising therapeutic strategy for MPM. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  18. Tumor promoters alter the temporal program of adenovirus replication in human cells.

    Science.gov (United States)

    Fisher, P B; Young, C S; Weinstein, I B; Carter, T H

    1981-04-01

    In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

  19. DNA transformations of Candida tropicalis with replicating and integrative vectors.

    Science.gov (United States)

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

  20. In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

    Institute of Scientific and Technical Information of China (English)

    Dennis Hoffmann; Wibke Bayer; Oliver Wildner

    2007-01-01

    AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and "F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/ F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector-treated tumor. We assessed the induction of a tumor-specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay.RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

  1. Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models.

    Science.gov (United States)

    Staba, M J; Wickham, T J; Kovesdi, I; Hallahan, D E

    2000-01-01

    Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.

  2. A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication.

    Science.gov (United States)

    Fechner, Henry; Wang, Xiaomin; Picó, Almudena Hurtado; Wildner, Judith; Suckau, Lennart; Pinkert, Sandra; Sipo, Isaac; Weger, Stefan; Poller, Wolfgang

    2007-01-20

    Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.

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

    Directory of Open Access Journals (Sweden)

    El Andaloussi Samir

    2011-05-01

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

  4. Effect of adenovirus gene transfer vectors on the immunologic functions of mouse dendritic cells.

    Science.gov (United States)

    Korst, Robert J; Mahtabifard, Ali; Yamada, Reiko; Crystal, Ronald G

    2002-03-01

    To address the effect of adenovirus (Ad) gene transfer vector transduction on the diverse functions of dendritic cells, we used an Ad vector encoding no transgene (AdNull) to transduce mouse bone-marrow-derived dendritic cells (BMDC). Initial experiments using an Ad vector encoding a marker gene (AdGFP, jellyfish green fluorescent protein) showed that the optimal ratio of infectious Ad particles to each cell was 100, when both transgene expression and resultant BMDC viability were taken into account. Exposure to AdNull resulted in upregulation of both surface activation markers (CD40, MHC class II, B7.1, B7.2, ICAM-1) and IL-12 expression by BMDC. AdNull activation of BMDC was observed in multiple strains of mice. Despite this, AdNull-transduced BMDC displayed only modestly impaired antigen uptake ability, as demonstrated in macropinocytosis and phagocytosis assays, in vitro. However, Ad-modified BMDC migrated to regional lymph nodes five times more efficiently than sham-transduced BMDC in vivo. In addition, Ad transduction significantly enhanced the ability of BMDC to present a model peptide antigen to T-lymphocyte hybridoma cells at low BMDC:T cell ratios. We conclude that Ad modification, in and of itself, induces a state of activation in mouse BMDC. This activation, albeit mild compared with that induced by other stimuli, produces measurable effects of the specific immunological functions of these antigen-presenting cells.

  5. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui

    2011-01-01

    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

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

    Science.gov (United States)

    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.

  7. Intravenous administration of the conditionally replicative adenovirus Ad5-Δ24RGD induces regression of osteosarcoma lung metastases

    Directory of Open Access Journals (Sweden)

    Gerritsen Winald R

    2008-01-01

    Full Text Available Abstract Metastatic osteosarcoma (OS has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Δ24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Δ24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Δ24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1–3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Δ24RGD-treated mice (p = 0.15. Moreover, mice treated at weeks 5–7 showed a significantly reduced lung weight (decrease of tumor mass, p 149, p = 0.12 compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Δ24RGD might be a promising new treatment strategy for metastatic osteosarcoma.

  8. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

    Science.gov (United States)

    Worgall, Stefan; Krause, Anja; Rivara, Michael; Hee, Kyung-Kim; Vintayen, Enrico V; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G

    2005-05-01

    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

  9. A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

    Science.gov (United States)

    Ching, Wilhelm; Koyuncu, Emre; Singh, Sonia; Arbelo-Roman, Christina; Hartl, Barbara; Kremmer, Elisabeth; Speiseder, Thomas; Meier, Chris; Dobner, Thomas

    2013-03-01

    Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

  10. A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

    Directory of Open Access Journals (Sweden)

    Wilhelm Ching

    2013-03-01

    Full Text Available Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

  11. Expression of biologically active atrial natriuretic factor following intrahepatic injection of a replication-defective adenoviral vector in dogs.

    Science.gov (United States)

    Chetboul, V; Adam, M; Deprez, I; Ambriovic, A; Rosenberg, D; Crespeau, F; Saana, M; Pham, I; Eloit, M; Adnot, S; Pouchelon, J L

    1999-01-20

    Atrial natriuretic factor (ANF) is a potent natriuretic, diuretic, and vasoactive hormone produced and released by atrial cardiomyocytes. We investigated whether adenovirus-mediated ANF gene delivery to dogs leads to a sustained increase in circulating ANF levels resulting in long-lasting biological effects. An adenoviral vector containing the canine ANF cDNA under the control of the Rous sarcoma virus 3' long terminal repeat (AdRSV-ANF) was injected via the intrahepatic route to nonvaccinated 2-month-old dogs. In the first group of four dogs injected with AdRSV-ANF (10(10.2) TCID50), a short-lived increase in plasma ANF concentrations not associated with biological effects occurred 8-10 days after the injection, as compared with four control dogs injected with an adenovirus encoding a luciferase reporter gene (AdRSV-luc). In a second series of experiments, six dogs received AdRSV-ANF at a dose of 10(10) TCID50 and a replication-defective type 5 adenovirus harboring a modified VAI gene (Ad-VAr) at the same dose. Sustained increases in plasma ANF concentrations and urinary cGMP excretion starting on day 2 and persisting until day 20 were seen, as well as concomitant elevations in natriuresis and diuresis, a transient increase in cardiac output, and a delay in body weight gain, as compared with control dogs injected with AdRSV-luc/Ad-VAr. These results show that adenovirus-mediated ANF gene expression can lead to systemic biological effects in dogs, a finding of potential relevance for the treatment of cardiovascular diseases and sodium-retaining disorders.

  12. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    Science.gov (United States)

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C

    1998-01-01

    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  13. Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

    Institute of Scientific and Technical Information of China (English)

    Xin-Bo Xue; Kun Chen; Cong-Jun Wang; Jian-Wei Zheng; Yuan Yu; Zhi-Hai Peng; Zai-De Wu

    2008-01-01

    BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modiifed Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was conifrmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and lfow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P CONCLUSIONS: mda-7 selectively induces growth inhibi-tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

  14. Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

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    Farley Daniel C

    2010-12-01

    Full Text Available Abstract Background Adenovirus serotype 5 (Ad5 has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be

  15. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    Science.gov (United States)

    Wigand, R

    1979-01-01

    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  16. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    Science.gov (United States)

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  17. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    Science.gov (United States)

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

  18. Molecular basis for viral selective replication in cancer cells: activation of CDK2 by adenovirus-induced cyclin E.

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    Full Text Available Adenoviruses (Ads with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL, promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

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

    Science.gov (United States)

    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.

  20. Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus

    Indian Academy of Sciences (India)

    Helin Li; Pengbo Ning; Zhi Lin; Wulong Liang; Kai Kang; Lei He; Yanming Zhang

    2015-03-01

    The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×106 TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

  1. The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

    Directory of Open Access Journals (Sweden)

    Jasdave S Chahal

    Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

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

    Science.gov (United States)

    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.

  3. Functional interactions of antiapoptotic proteins and tumor necrosis factor in the context of a replication-competent adenovirus.

    Science.gov (United States)

    Liu, T-C; Wang, Y; Hallden, G; Brooks, G; Francis, J; Lemoine, N R; Kirn, D

    2005-09-01

    Replication-selective oncolytic adenoviruses hold promise, but novel mechanisms must be identified to maximize intratumoral virus persistence, spread and therapeutic transgene-carrying capacity while maintaining safety. One of the main approaches to engineering cancer-selectivity has been to delete a viral gene that is theoretically expendable in cancer cells. Results with this approach have been mixed, however, as evidenced by controversy over Onyx-015 (E1B-55kD(-)) selectivity. We hypothesized that the functional redundancy between viral gene products might limit selectivity and/or potency with this approach. Antiviral immune inducers of apoptosis (eg TNF-alpha) have not been thoroughly investigated in previous studies. We therefore explored whether deletion of functionally redundant viral genes, E1B-19kD and E3B, both independently antagonize TNF-alpha, could lead to enhanced oncolytic potency while maintaining selectivity. Since tumors have numerous blocks in apoptotic pathways, we hypothesized that deletion of one or both gene regions would result in cancer-selectivity in the presence of TNF-alpha. We have previously shown that the E1B-19kD deletion resulted in enhanced viral spread in vitro and in immunocompetent tumor models in vivo. In contrast, the impact of E3B deletion, especially its in vitro selectivity and potency, was not thoroughly characterized, although it resulted in rapid immune-mediated viral clearance in vivo. Furthermore, previous publications indicated that double-deleted mutants have selectivity but unsatisfactory efficacy. We compared the selectivity and potency of E1B-19kD(-), E3B(-) and E1B-19kD(-)/E3B(-) mutants to wild-type adenovirus. In cancer cells, the E1B-19kD(-) mutant had superior replication, spread and cytolysis (+) or (-) TNF-alpha; deletion of both E1B-19kD and E3B was relatively deleterious. In normal cells without TNF-alpha, similar results were obtained. In contrast, all three mutants were significantly inhibited in the

  4. A Regulatory Element Near the 3′ End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts

    Science.gov (United States)

    Hammer, Eva; Gonsior, Melanie; Stutika, Catrin; Heilbronn, Regine

    2016-01-01

    ABSTRACT Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3′ end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RIS-Ad). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5′ half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCE Insertion of sequences from the 3′ part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so

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

    Directory of Open Access Journals (Sweden)

    Johanna P Laakkonen

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

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

    Directory of Open Access Journals (Sweden)

    Wenli Zhang

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat......, vif, rev and vpr antigens fused to the MHC class II associated invariant chain. Immunizations induced broad T cell responses in all vaccinees. Following up to 10 repeated low-dose intrarectal challenges, vaccinees suppressed early viral replication (P=0.01) and prevented the peak viremia in 5....../6 animals. Despite consistently undetectable viremia in 2 out of 6 vaccinees, all animals showed evidence of infection induced immune responses indicating that infection had taken place. Vaccinees, with and without detectable viremia better preserved their rectal CD4+ T cell population and had reduced...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  9. Midkine promoter-driven suicide gene expression and -mediated adenovirus replication produced cytotoxic effects to immortalised and tumour cells.

    Science.gov (United States)

    Yu, L; Hamada, K; Namba, M; Kadomatsu, K; Muramatsu, T; Matsubara, S; Tagawa, M

    2004-07-01

    We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

  10. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells: not as bad as their reputation.

    Science.gov (United States)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang; Jensen, Benjamin Anderschou Holbech; Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2013-06-01

    It has been reported that adenovirus (Ad)-primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells by directly comparing these cells to CD8 T cells induced through infection with lymphocytic choriomeningitis virus (LCMV). We found that localized immunization with intermediate doses of Ad vector induces a moderate number of functional CD8 T cells which qualitatively match those found in LCMV-infected mice. The numbers of these cells may be efficiently increased by additional adenoviral boosting, and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad vaccination led to even higher numbers of memory cells. In this case, the vaccination led to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression. These memory CD8 T cells were capable of proliferating in response to viral challenge and protecting against infection with live virus. Furthermore, viral challenge was followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells did not appear to have been driven toward exhaustive differentiation. Based on these findings, we suggest that adenovirus-based prime-boost regimens (including Ad serotype 5 [Ad5] and Ad5-like vectors) represent an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells.

  11. Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

    Institute of Scientific and Technical Information of China (English)

    XIN LEI LIU; SHUNAG QING YU; XIA FENG; XIAO LI WANG; HONG MEI LIU; XIAO MEI ZHANG; HONG XIA LI; LING ZHOU; YI ZENG

    2006-01-01

    The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod. gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod. gag, rAd5-mod. gag or DNA and were boosted after 3 weeks.To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIVspecific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

  12. Tamoxifen-regulated adenoviral E1A chimeras for the control of tumor selective oncolytic adenovirus replication in vitro and in vivo.

    Science.gov (United States)

    Sipo, I; Wang, X; Hurtado Picó, A; Suckau, L; Weger, S; Poller, W; Fechner, H

    2006-01-01

    Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E(DeltaNLS)M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E(DeltaNLS)M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.E(DeltaNLS)M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E(DeltaNLS)M and Ad.MEM in vivo. Induction of Ad.E(DeltaNLS)M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E(DeltaNLS)M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.

  13. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Science.gov (United States)

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P

    1998-03-01

    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  14. Ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro.

    Science.gov (United States)

    Hartline, Caroll B; Gustin, Kortney M; Wan, William B; Ciesla, Stephanie L; Beadle, James R; Hostetler, Karl Y; Kern, Earl R

    2005-02-01

    The acyclic nucleoside phosphonate cidofovir (CDV) and its closely related analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA) have been reported to have activity against many adenovirus (AdV) serotypes. A new series of orally active ether lipid-ester prodrugs of CDV and of (S)-HPMPA that have slight differences in the structure of their lipid esters were evaluated, in tissue-culture cells, for activity against 5 AdV serotypes. The results indicated that, against several AdV serotypes, the most active compounds were 15-2500-fold more active than the unmodified parent compounds and should be evaluated further for their potential to treat AdV infections in humans.

  15. The effect of purine and pyrimidine analogues and virazole on adenovirus replication.

    Science.gov (United States)

    Scheffler, P; Haghchenas, D; Wigand, R

    1975-04-01

    The multiplication of adenovirus 19 in HeLa cells was inhibited by various purine and pyrimidine analogues and by virazole. The formation of infectious virus and of capsid proteins (haemagglutin, group-specific complement-fixing antigen) was inhibited to the same degree, while the viral cytopathic effect (CPE) was not inhibited. The reversibility of the inhibition after removal of the substances was more complete for purine than for pyrimidine analogues. The inhibition was counteracted by simulataneous addition of the corresponding nucleosides. Adenosine was more effected than guanosine against purine analogues; both were partially effective against virazole, but none of them against arabinofuranosyladenine. The time-dependence of inhibition, the ensuing eclipse period after removal of the inhibitors, and the successive application of two inhibitors led to the conclusion that most of them affect the viral multiplication mainly by inhibition of DNA synthesis. Azacytidine inhibits the synthesis of structural proteins as well.

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

    Science.gov (United States)

    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

  17. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Directory of Open Access Journals (Sweden)

    Frank Sainsbury

    Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

  18. Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

    Institute of Scientific and Technical Information of China (English)

    刘启功; 陆再英; 岳远坤; 林立; 张卫东; 颜进

    2004-01-01

    This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer.The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

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

    2005-12-20

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

  1. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 2'-nor-cyclic GMP on adenovirus replication in vitro.

    Science.gov (United States)

    Baba, M; Mori, S; Shigeta, S; De Clercq, E

    1987-02-01

    The inhibitory effects of 20 selected antiviral compounds on the replication of adenoviruses (types 1 to 8) in vitro were investigated. While 18 compounds were ineffective, 2 compounds, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cyclic GMP), were highly effective against all adenovirus types assayed in human embryonic fibroblast cultures. Their 50% inhibitory doses were 1.1 microgram/ml for (S)-HPMPA and 4.1 micrograms/ml for 2'-nor-cyclic GMP. They were nontoxic for the host cells at the effective antiviral doses.

  2. Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.

    Science.gov (United States)

    Merentie, M; Lottonen-Raikaslehto, L; Parviainen, V; Huusko, J; Pikkarainen, S; Mendel, M; Laham-Karam, N; Kärjä, V; Rissanen, R; Hedman, M; Ylä-Herttuala, S

    2016-03-01

    Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

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

    Science.gov (United States)

    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.

  4. Properties and use of novel replication-competent vectors based on Semliki Forest virus

    Directory of Open Access Journals (Sweden)

    Lulla Valeria

    2009-03-01

    Full Text Available Abstract Background Semliki Forest virus (SFV has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. Results We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. Conclusion The properties of replication-competent vectors

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-05

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

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

    Directory of Open Access Journals (Sweden)

    Shunsuke Iizuka

    2017-09-01

    Full Text Available 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.

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

    Science.gov (United States)

    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 (PBMP2 mRNA between group A and C (PBMP2 in group A and C was markedly higher than that in groups B, D and E (PBMP2-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.

  10. Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

    Science.gov (United States)

    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

    2004-03-01

    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 beta-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 beta-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 beta-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 beta-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-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-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 AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-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 data demonstrate that addition of an RGD motif

  11. Expanding specificity of class I restricted CD8+ T cells for viral epitopes following multiple inoculations of swine with a human adenovirus vectored foot-and-mouth disease virus (FMDV) vaccine

    DEFF Research Database (Denmark)

    Pedersen, Lasse E.; Patch, Jared R; Kenney, Mary

    2016-01-01

    The immune response to the highly acute foot-and-mouth disease virus (FMDV) is routinely reported as a measure of serum antibody. However, a critical effector function of immune responses combating viral infection of mammals is the cytotoxic T lymphocyte (CTL) response mediated by virus specific CD......8 expressing T cells. This immune mechanism arrests viral spread by killing virus infected cells before new, mature virus can develop. We have previously shown that infection of swine by FMDV results in a measurable CTL response and have correlated CTL killing of virus-infected cells with specific...... class I major histocompatibility complex (MHC) tetramer staining. We also showed that a modified replication defective human adenovirus 5 vector expressing the FMDV structural proteins (Ad5-FMDV-T vaccine) targets the induction of a CD8(+) CTL response with a minimal humoral response. In this report, we...

  12. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G., E-mail: chinnag@slu.edu

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.

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

    Science.gov (United States)

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

    2012-01-01

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

  14. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  15. Transgene stability for three replication-competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Duch, Mogens R.; Carrasco, Maria L; Hansen, Bettina Dencker

    2004-01-01

    Retroviral vectors that are able to sustain multiple rounds of replication may find many applications. However, one critical feature of such vectors is the ability to maintain an intact transgene cassette during repeated rounds of replication. We here report on the stability of a translational...... cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third...

  16. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Institute of Scientific and Technical Information of China (English)

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral

  17. Efficient induction of cross-presentating human B cell by transduction with human adenovirus type 7 vector.

    Science.gov (United States)

    Peng, Ying; Lai, Meimei; Lou, Yunyan; Liu, Yanqing; Wang, Huiyan; Zheng, Xiaoqun

    2016-01-01

    Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8(+) T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8(+) T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy.

  18. Antigen Gene Transfer to Human Plasmacytoid Dendritic Cells Using Recombinant Adenovirus and Vaccinia Virus Vectors

    Directory of Open Access Journals (Sweden)

    Hetty J. Bontkes

    2005-01-01

    Full Text Available Recombinant adenoviruses (RAd and recombinant vaccinia viruses (RVV expressing tumour-associated antigens (TAA are used as anti-tumour vaccines. It is important that these vaccines deliver the TAA to dendritic cells (DC for the induction of a strong immune response. Infection of myeloid DC (MDC with RAd alone is relatively inefficient but CD40 retargeting significantly increases transduction efficiency and DC maturation. Infection with RVV is efficient without DC maturation. Plasmacytoid dendritic cells (PDC play a role in the innate immune response to viral infections through the secretion of IFNα but may also play a role in specific T-cell induction. The aim of our study was to investigate whether PDC are better targets for RAd and RVV based vaccines. RAd alone hardly infected PDC (2% while CD40 retargeting did not improve transduction efficiency, but it did increase PDC maturation (25% CD83 positive cells. Accordingly, specific CTL activation by RAd infected PDC was limited (the number of IFNγ producing CTL was reduced by 75% compared to stimulation with peptide loaded PDC. RVV infected PDC specifically stimulated CTL but PDC were not activated. These Results indicate that PDC are not ideal targets for RAd and RVV based vaccines. However, PDC induced specific CTL activation after pulsing with recombinant protein, indicating that PDC can also cross-present antigens released from surrounding infected cells.

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

    Science.gov (United States)

    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.

  20. Inhibition of avian leukosis virus replication by vector-based RNA interference

    Science.gov (United States)

    RNAi has recently emerged as a promising antiviral technique in vertebrates. To date, most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, though vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are...

  1. An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

    Directory of Open Access Journals (Sweden)

    Magalhães GS

    2002-01-01

    Full Text Available Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK gene, followed by administration of the antiviral drug ganciclovir (GCV. The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK driven by three strong promoters (P CMV IE, SV40 and EN1 and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

  2. Replication of Tomato Yellow Leaf Curl Virus in Its Whitefly Vector, Bemisia tabaci.

    Science.gov (United States)

    Pakkianathan, Britto Cathrin; Kontsedalov, Svetlana; Lebedev, Galina; Mahadav, Assaf; Zeidan, Muhammad; Czosnek, Henryk; Ghanim, Murad

    2015-10-01

    Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted exclusively by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial, and thus far, the virus has been considered to be nonpropagative. Following 8 h of acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (>2-fold) during the first few days and then continuously decreased, as measured by the amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on whiteflies, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching of whiteflies between pesticide, heat stress, and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector. Plant viruses in agricultural crops are of great concern worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within these vectors. Such replication is important, as it contributes to the

  3. Relationships between resistance to cross-linking agents and glutathione metabolism, aldehyde dehydrogenase isozymes and adenovirus replication in human tumour cell lines.

    Science.gov (United States)

    Parsons, P G; Lean, J; Kable, E P; Favier, D; Khoo, S K; Hurst, T; Holmes, R S; Bellet, A J

    1990-12-15

    In a panel of 10 human tumour cell lines with no prior exposure to drugs in vitro, resistance to cisplatin correlated with resistance to the nitrogen mustard derivatives Asta Z-7557 (mafosfamide, an activated form of cyclophosphamide), melphalan and chlorambucil. Simultaneous treatment with DL-buthionine-S,R-sulfoximine did not enhance the toxicity of cisplatin or Asta Z-7557, and no correlation was found between drug resistance and cellular levels of metallothioneins (as judged by sensitivity to cadmium chloride), glutathione (GSH), GSH reductase, GSH transferase, or gamma-glutamyltranspeptidase. The two cell lines most resistant to Asta Z-7557 expressed aldehyde dehydrogenase cytosolic isozyme 1, found also in normal ovary, but not isozyme 3. Treatment of resistant cells with cisplatin or Asta Z-7557 inhibited cellular DNA synthesis and replication of adenovirus 5 to a lesser extent than in sensitive cells. The virus could be directly inactivated by both drugs prior to infection, subsequent replication being inhibited to the same extent in sensitive and resistant cells. In contrast to Asta Z-7557 and other DNA damaging agents, cisplatin was much more toxic to adenovirus (D37 0.022-0.048 microM) than to cells (D37 0.25-2.5 microM). The adenovirus 5 mutant Ad5ts125 having a G----A substitution was even more sensitive to cisplatin (D37 7-8 nM) than wild type virus and another mutant. Cisplatin was detoxified less by sonicated resistant resistant cells than sensitive cells, as judged by inactivation of Ad5ts125 added to the reaction mixture. It can be inferred that (i) the major differences in cellular resistance to cisplatin and Asta Z-7557 in the present material did not involve enhanced DNA repair or protection by metallothioneins or GSH, but were associated with the ability to continue cellular and viral DNA synthesis during treatment, (ii) resistance was not associated with less template damage, and (iii) the adenovirus genome may be a suitable probe for

  4. CXCR4启动子的条件复制型腺病毒对肺癌细胞的靶向杀伤作用%Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

    Institute of Scientific and Technical Information of China (English)

    李龙光; 李书华; 王红艳; 龙捷; 谢晓斌; 张雅洁

    2015-01-01

    [ ABSTRACT] AIM:To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically.METHODS:Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP.The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP.PCR was used to detect the target gene fragments, and the viral titer was determined.A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR.CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL.CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay.RE-SULTS:The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully.Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus.PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome.The virus in the supernatant reached a titer of 1 ×1013 PFU/L.The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group.Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group

  5. 大鼠乙醛脱氢酶2基因调控腺病毒载体构建方法及意义%Methodology on construction of rat aldehyde dehydrogenase 2 gene regulation recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    李鸿博; 郎小娥

    2014-01-01

    目的:观察持续活化突变体腺病毒转染大鼠心肌细胞的转染效果及对乙醛脱氢酶2(ALDH2)表达的影响。方法分别将扩增得到的 ALDH2持续活化突变体基因及合成 ALDH2-siRNA 序列颈环状 DNA,连接相应载体后,得到重组穿梭质粒;对2种穿梭质粒分别进行扩增和酶切鉴定,并导入 pAdeno 腺病毒载体,转染293细胞进行扩增与纯化。将1日龄雄性 SD 大鼠心肌细胞进行培养,将2种重组腺病毒及对照空载体分别感染细胞,随后检测 ALDH2表达量。结果2种载体构建正确,纯化后两者滴度分别为2×1010,1.6×1010 PFU・ mL-1。实验组 ALDH2表达量与对照组相比差异具有统计学意义(P <0.01)。结论成功构建大鼠 ALDH2基因双向调控腺病毒载体,可以有效调控离体大鼠心肌细胞 ALDH2表达。%Objective To construct adenovirus specific for rat aldehyde dehydrogenase 2 (ALDH2) gene interference and consistent activation and transfect the viruses into rat cardiomyocytes to observe transfection effect and its influence on ALDH2 expression.Methods Consistently active ALDH2 (CA -ALDH2) mutant gene was amplified and linked to shuttle vector, thus recombinant shuttle plasmid was subsequently con -structed.Stem -loop DNA for ALDH2 silencing RNA ( ALDH2 -siRNA) sequence was synthesized and loaded it into vector thus recombi -nant shuttle plasmid was constructed .Both kinds of plasmid were imple -mented amplification and enzyme identification.Verified plasmids were loaded into pAdeno adenovirus vectors.The viruses were then transfected into 293 cell linage to replicate and be purified.Treat cultured cardio-myocytes from 1 -day -old neonatal male Sprague Dawley (SD) rat with empty adenovirus vector control and both kinds of recombinant adenovirus vector, and perform subsequent assay for ALDH2 expression.Results Both vectors are identified by endonuclease with titre of 2 ×10 10 , 1.6 ×10 10 PFU ・ mL-1

  6. Adenovirus structure.

    Science.gov (United States)

    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.

  7. Acceleration of adenovirus replication and increased virion production by treatment with the steroid hormone 17 beta-estradiol.

    Science.gov (United States)

    James, C B; Vanderpool, E A; Roane, P

    1992-01-01

    We report here that concentration of an estrogen known to promote enhanced transformation and to increase oncogenicity of rat embryo cells, accelerate the production and increase the yield of progeny virions in adenovirus type 12 (Ad 12)-infected HEp-2 cells. Further, measurement of the incorporation of radioactive RNA and DNA precursors indicated that macromolecular synthesis in the estrogen-treated, infected cells was accelerated. Possible explanations for this observation are discussed.

  8. Enhanced antitumoral efficacy and immune response following conditionally replicative adenovirus containing constitutive HSF1 delivery to rodent tumors

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    Fan Rong

    2012-05-01

    Full Text Available Abstract Background Oncolytic adenoviruses are promising as anticancer agents but have limited clinical responses. Our previous study showed that heat shock transcription factor 1 (HSF1 overexpression could increase the anti-tumor efficacy of E1B55kD deleted oncolytic adenovirus through increasing the viral burst. Due to the important roles of heat shock proteins (HSPs in eliciting innate and adaptive immunity, we reasoned that besides increasing the viral burst, HSF1 may also play a role in increasing tumor specific immune response. Methods In the present study, intra-dermal murine models of melanoma (B16 and colorectal carcinoma (CT26 were treated with E1B55kD deleted oncolytic adenovirus Adel55 or Adel55 incorporated with cHSF1, HSF1i, HSP70, or HSP90 by intra-tumoral injection. Tumors were surgically excised 72 h post injection and animals were analyzed for tumor resistance and survival rate. Results Approximately 95% of animals in the Adel55-cHSF1 treated group showed sustained resistance upon re-challenge with autologous tumor cells, but not in PBS, Adel55, or Adel55-HSF1i treated groups. Only 50–65% animals in the Adel55-HSP70 and Adel55-HSP90 treated group showed tumor resistance. Tumor resistance was associated with development of tumor type specific cellular immune responses. Adel55-cHSF1 treatment also showed higher efficacy in diminishing progression of the secondary tumor focus than Adel55-HSP70 or Adel55-HSP90 treatment. Conclusions Besides by increasing its burst in tumor cells, cHSF1 could also augment the potential of E1B55kD deleted oncolytic adenovirus by increasing the tumor-specific immune response, which is beneficial to prevent tumor recurrence. cHSF1 is a better gene for neoadjuvant immunotherapy than other heat shock protein genes.

  9. Chapter three--Syrian hamster as an animal model to study oncolytic adenoviruses and to evaluate the efficacy of antiviral compounds.

    Science.gov (United States)

    Wold, William S M; Toth, Karoly

    2012-01-01

    The Syrian (golden) hamster (Mesocricetus auratus) has served as a useful model for different aspects of biology for at least 50 years, and its use has been expanding recently. In earlier years, among other things, it was a model for cancer development. More recently, it has become a model for many different infectious diseases. It has also become an alternative model for the study of oncolytic adenovirus vectors for cancer gene therapy. Among several other human pathogens, the hamster is permissive for the replication of human species C adenoviruses, which are the parental virus for the majority of adenovirus vectors in use today. These vectors replicate in some of the established hamster tumor cell lines that can be used to generate tumors in vivo, that is, one can study oncolytic (replication competent) adenoviruses in a permissive, immunocompetent model. This has afforded the opportunity to study the effect of the host immune system on the vector-infected tumor and has allowed the use of a more relevant animal model to determine the safety and biodistribution of replication-competent adenoviruses. The hamster has also been used to evaluate antiviral compounds and vaccines against many viruses, including adenoviruses, flaviviruses, alphaviruses, arenaviruses, bunyaviruses, and paramyxoviruses.

  10. Engineering cell lines for production of replication defective HSV-1 gene therapy vectors.

    Science.gov (United States)

    Grant, Kyle G; Krisky, David M; Ataai, Mohammed M; Glorioso, Joseph C

    2009-03-01

    Herpes simplex virus type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Our vectors are deleted for the essential immediate early genes ICP4 and ICP27. These genes are controlled by unique promoters having enhancer elements responsive to a viral structural protein VP16. The expression of these genes occurs prior to the activation of all other lytic functions and is thus required to initiate and complete the virus replication cycle. For large scale manufacture of clinical grade vectors, efficient cell lines must be generated that express the essential viral gene products in trans during vector propagation. Here we describe methods for engineering HSV-1 production cell lines that improve vector growth by altering the kinetics of complementing gene expression. We examined the ability of Vero cells independently transduced with ICP4 and ICP27 under transcriptional control of their respective promoters to support the growth of a replication defective vector (JDTOZHE), deleted for ICP4, ICP27 and approximately 20 kb of internal elements that are not required for virus growth in Vero cells. Vector yield on this cell line was 3 logs lower than wild-type virus grown on Vero cells. To understand the mechanism underlying poor vector yield, we examined the expression of ICP4 and ICP27 during virus complementation. While ICP27 was expressed immediately on vector infection, the expression of ICP4 was considerably delayed by 8-10 h, suggesting that the ICP4 promoter was not adequately activated by VP16 delivered by the infectious vector particle. Use of the ICP0 promoter to express ICP4 from the cellular genome resulted in higher induction levels and faster kinetics of ICP4 expression and a 10-fold improvement in vector yield. This study suggests that vector complementation is highly dependent on the

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

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    Emma-Jo Hayton

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

  12. Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors.

    Science.gov (United States)

    Sgourou, Argyro; Routledge, Samantha; Spathas, Dionysios; Athanassiadou, Aglaia; Antoniou, Michael N

    2009-08-20

    Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

  13. Enhanced immunity against classical swine fever in pigs induced by prime-boost immunization using an alphavirus replicon-vectored DNA vaccine and a recombinant adenovirus.

    Science.gov (United States)

    Sun, Yuan; Li, Na; Li, Hong-Yu; Li, Miao; Qiu, Hua-Ji

    2010-09-15

    Classical swine fever (CSF) - caused by the classical swine fever virus (CSFV) - is a fatal disease of pigs that is responsible for extensive losses to the swine industry worldwide. We had demonstrated previously that a prime-boost vaccination strategy using an alphavirus (Semliki Forest virus, SFV) replicon-vectored DNA vaccine (pSFV1CS-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV induced enhanced immune responses in a mouse model. In this study, we evaluated further the efficacy of the heterologous prime-boost immunization approach in pigs, the natural host of CSFV. The results showed that the pigs (n=5) receiving pSFV1CS-E2/rAdV-E2 heterologous prime-boost immunization developed significantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4(+) and CD8(+) T-cell proliferation, compared to the pigs receiving double immunizations with rAdV-E2 alone. When challenged with virulent CSFV Shimen strain, the pigs of the heterologous prime-boost group did not show clinical symptoms or viremia, which were observed in one of the 5 pigs immunized with rAdV-E2 alone and all the 5 control pigs immunized with an empty adenovirus. The results demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy can induce solid protective immunity.

  14. A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

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    Michael C Keefer

    Full Text Available 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 2×10(9 (A, 2×10(10 (B, 2×10(11 (C, or Ad35-GRIN 1×10(10 (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 10(6 PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. 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

  15. E4orf6 variants with separate abilities to augment adenovirus replication and direct nuclear localization of the E1B 55-kilodalton protein.

    Science.gov (United States)

    Orlando, Joseph S; Ornelles, David A

    2002-02-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

  16. Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland†

    Science.gov (United States)

    Zheng, Changyu; Nikolov, Nikolay P.; Alevizos, Ilias; Cotrim, Ana P.; Liu, Shuying; McCullagh, Linda; Chiorini, John A.; Illei, Gabor G.; Baum, Bruce J.

    2017-01-01

    Background Radiation-induced salivary hypofunction is a common side-effect of treatment for head and neck cancers. Patients suffer significant morbidity and there is no suitable conventional therapy. We are conducting a Phase I clinical trial, using a first-generation serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) to treat such patients. One week after the administration of AdhAQP1 to an enrolled, generally healthy patient, E1-containing adenovirus was detected in parotid saliva. Methods The real-time quantitative polymerase chain reactuion (PCR) was used to measure the Ad5 E1 gene and AdhAQP1 in saliva and serum. PCR and sequencing were used to characterize viral/vector DNA extracted from saliva. The presence of infectious adenovirus was assessed by the inoculation of A549 cells with aliquots of saliva. Serum Ad5 neutralizing antibodies were measured by the inhibition of 293-cell transduction with an Ad5 vector encoding luciferase. Multiple clinical evaluations were performed. Results On day 7 after AdhAQP1 delivery, low levels of the Ad5 E1 gene were detected in parotid saliva (82 copies/μl). In addition, significant levels of AdhAQP1 were also detected (1.5 × 103 copies/μl). The patient was asymptomatic and subsequent analysis of parotid saliva samples prior to day 7 and after day 7 until day 42 was negative for both virus and vector. No virus or vector was detected in serum at any time. Detailed PCR analyses of DNA extracted from the day 7 parotid saliva sample suggested the absence of a recombination event, and no infectious virus was found. Conclusions The patient most likely had a latent Ad5 infection in the targeted parotid gland that was activated after gene transfer and was without clinical consequence. Published in 2009 by John Wiley & Sons, Ltd. PMID:19941317

  17. Adenovirus 5-vectored P. falciparum vaccine expressing CSP and AMA1. Part A: safety and immunogenicity in seronegative adults.

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    Martha Sedegah

    Full Text Available BACKGROUND: Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers. METHODOLOGY/PRINCIPAL FINDINGS: The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. Group 1 (n = 6 healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct and Group 2 (n = 6 a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045 group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003, but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7-10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites. SIGNIFICANCE: As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a

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

    Science.gov (United States)

    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.

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

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    Daniel C Farley

    2015-01-01

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

  20. Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus

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    Fuminori Sakurai

    2016-01-01

    Full Text Available Circulating tumor cells (CTCs are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP–expressing conditionally replicating adenovirus (Ad (rAd-GFP was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus–adenovirus receptor (CAR cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell–specific microRNA, miR-142-3p, were incorporated into the 3′-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.

  1. ADENOVIRUS-MEDIATED WILD-TYPE P53 EXPRESSION SUPPRESSES GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji

    1998-01-01

    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  2. Construction and Expression of Recombinant Adenovirus Vector Ad5-hTRX-EGFP%重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

    Institute of Scientific and Technical Information of China (English)

    扈江伟; 王军; 徐曼; 苏永锋; 孔维霞; 盛红霞; 张斌; 陈虎

    2012-01-01

    本研究旨在构建并制备人硫氧还蛋白(hTRX)和增强型绿色荧光蛋白(EGFP)基因腺病毒载体Ad-hTRX-EGFP,感染HEK293细胞,为基因治疗提供实验基础.设计含有Not Ⅰ和EcoR Ⅴ酶切位点的引物,PCR扩增hTRX,将扩增产物连接到带有EGFP标记的pDC316-mCMV穿梭质粒上,构建重组穿梭质粒pDC316-hTRX-EG-FP,利用Lipofectamine2000脂质体的方法将AdMax腺病毒包装系统的骨架质粒pBHG lox_E1,3Cre和穿梭质粒pDC316-hTRX-EGFP共转染入HEK293细胞,进行同源重组,得到腺病毒重组质粒pAd-hTRX-EGFP,并在其中包装扩增病毒,用氯化铯高速梯度离心、纯化病毒,测定病毒颗粒数及滴度.采用PCR方法对重组腺病毒进行鉴定,用流式细胞仪测定感染HEK293细胞的效率,Western blot方法验证细胞表达hTRX蛋白.结果显示,重组腺病毒质粒经PCR和Not Ⅰ、EcoR Ⅴ酶切鉴定,证实含有hTRX基因,测序结果和设计片段的序列一致.重组腺病毒载体构建成功,病毒滴度达5.558 × 1010pfu/ml.病毒成功感染HEK293细胞,MOI=100时,感染效率达92.25%.经Western blot方法验证表明,感染后的HEK293细胞高表达hTRX蛋白.结论:应用细胞内同源重组方法成功构建了含hTRX基因的重组腺病毒载体,制备获得高滴度的病毒,能高效感染HEK293细胞并表达目的蛋白,为后续研究奠定了基础.%This study was purposed to construct and prepare the recombinant adenovirus vector carrying human thioredoxin (hTRX) and enhanced green fluorescence protein ( EGFP), and transfect it into HEK293 cells,so as to lay a foundation for further gene therapy. The PCR-amplified products of hTRX with a pair of primers containing Not I and EcoR V restriction sites were subcloned into shuttle plasmid pDC316-mCMV. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and large adenovirus-helper plasmid pBHGlox( delta) El, 3Cre in mediation of liposome. The obtained replication

  3. 腺病毒介导的HSV-tk 体外抗喉癌作用%Anti-laryngeal carcinoma with adenovirus vector mediated HSV-tk gene

    Institute of Scientific and Technical Information of China (English)

    陈立; 李凡; 易世红; 黄红兰; 赵春燕; 魏强

    2001-01-01

    Objective To observe killing action of HSV-tk/GCV system onlaryngeal cancer cells. Methods Hep-2 cells were transfected by adenovirus vector with LacZ gene, then stained with X-gal for measurement of the adenovirus transfected efficiency. The recombinant adenovirus carrying HSV-tk was constructed by homologous recombination techniques. Hep-2 cells survival and by stander effect were observed after adding 10 mg/L GCV to transfected cells. Results The more the multiplicity of infection (MOI), the more the adenovirus transfected efficiency to Hep-2 cells. 100% transfected efficiency needs MOI 200. The killing action of HSV-tk/GCV on Hep-2 cells depends on the amount of AdCMVtk. There is a weak by stander effect. Conclusion Adenovirus mediated HSV-tk/GCV can kill Hep-2 cells.%目的 观察HSV-tk/GCV系统对喉癌细胞的杀伤作用。方法 将带有报告基因LacZ的5型缺陷性腺病毒载体AdCMVLacZ感染喉癌细胞Hep-2,X-gal染色,测定腺病毒的转染率。利用同源重组技术构建含HSV-tk基因的重组腺病毒载体AdCMVtk。AdCMVtk感染Hep-2细胞后,加入10mg/LGCV,观察瘤细胞存活率及旁观者效应。结果 随着腺病毒感染复数量(multiplicityofinfection,MOI)增加,对Hep-2细胞转染率亦增加,100%转染所需的MOI为200。AdCMVtk/GCV对Hep-2细胞的杀伤强度与AdCMVtk量呈正向关系,即有浓度依赖性。此杀伤作用存在旁观者效应,但较弱。结论 腺病毒介导的HSV-tk/GCV具有杀伤喉癌细胞作用。

  4. Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus.

    Science.gov (United States)

    Singh, Neetu; Pandey, Aseem; Jayashankar, Lakshmi; Mittal, Suresh K

    2008-05-01

    Because of the high prevalence of adenovirus (Ad) infections in humans, it is believed that pre-existing Ad-neutralizing antibodies (vector immunity) may negatively impact the immune response to vaccine antigens when delivered by human Ad (HAd) vectors. In order to evaluate whether bovine Ad subtype 3 (BAd3), a non-HAd vector, can effectively elude high levels of pre-existing vector immunity, naïve and HAd serotype 5 (HAd)-primed mice were immunized with BAd-H5HA [BAd3 vector expressing the hemagglutinin (HA) gene from H5N1 influenza virus]. Even in the presence of very high levels of HAd-specific neutralizing antibody, no significant reductions in HA-specific humoral and cell-mediated immune (CMI) responses were observed in HAd-primed mice immunized with BAd-H5HA. In naïve mice immunized with HAd-H5HA (HAd5 vector expressing H5N1 HA) and boosted with BAd-H5HA, the humoral responses elicited were significantly higher (P BAd-H5HA alone, while the CMI responses were comparable in the groups. This finding underlines the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of naïve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness.

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

    Science.gov (United States)

    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

  6. Canine recombinant adenovirus vector induces an immunogenicity-related gene expression profile in skin-migrated CD11b⁺ -type DCs.

    Directory of Open Access Journals (Sweden)

    Vanessa Contreras

    Full Text Available 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.

  7. The repression domain of the E1B 55-kilodalton protein participates in countering interferon-induced inhibition of adenovirus replication.

    Science.gov (United States)

    Chahal, Jasdave S; Gallagher, Courtney; DeHart, Caroline J; Flint, S J

    2013-04-01

    To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5'-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.

  8. Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component.

    Directory of Open Access Journals (Sweden)

    Cindy Tamminga

    Full Text Available BACKGROUND: A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. METHODOLOGY/PRINCIPAL FINDINGS: NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP. It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1 that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al. Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1 x 1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m] that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m. CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. SIGNIFICANCE: The NMRC-MV-Ad-PfC vaccine expressing CSP was

  9. A Dual-Action Armed Replicating Adenovirus for the Treatment of Osteoblastic Bone Metastases of Prostate Cancer

    Science.gov (United States)

    2007-03-01

    America, 91, 8802–8806. 69417. Chartier , C., Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A., 695& Mehtali, M. (1996). Efficient generation of...Biophysica Acta. 82847. Douglas, J. T., Rogers , B. E., Rosenfeld, M. E., Michael, S. I., 829Feng, M., & Curiel, D. T. (1996). Targeted gene delivery...by 830tropism-modified adenoviral vectors. Nature Biotechnology, 14, 8311574–1578. 83248. Dmitriev, I., Kashentseva, E., Rogers , B. E., Krasnykh, V

  10. The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

    Science.gov (United States)

    Danielsson, Angelika; Dzojic, Helena; Rashkova, Victoria; Cheng, Wing-Shing; Essand, Magnus

    2011-02-17

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

  11. The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

    Directory of Open Access Journals (Sweden)

    Angelika Danielsson

    Full Text Available The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR, leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

  12. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Directory of Open Access Journals (Sweden)

    Zihua Wang

    Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

  13. A modified hTERT promoter-directed oncolytic adenovirus replication with concurrent inhibition of TGFbeta signaling for breast cancer therapy.

    Science.gov (United States)

    Hu, Z; Robbins, J S; Pister, A; Zafar, M B; Zhang, Z-W; Gupta, J; Lee, K J; Newman, K; Neuman, K; Yun, C-O; Guise, T; Seth, P

    2010-04-01

    We were interested in developing oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we constructed mhTERTAd.sTbetaRFc, a 01/07-based adenoviral vector expressing the soluble form of transforming growth factor-beta (TGFbeta) receptor II fused with the human Fc IgG1 (sTGFbetaRIIFc) gene, in which viral replication is under the control of a modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTbetaRFc-mediated sTGFbetaRIIFc production targets the TGFbeta pathway known to contribute to the tumor progression of breast cancer metastasis. We chose to use the mhTERT promoter because it was found to be relatively more active (approximately 20 times) in breast cancer cells compared with normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 h with mhTERTAd.sTbetaRFc produced high levels of sTGFbetaRIIFc (greater than 1 microg ml(-1)) in the medium. Breast cancer cells produced nearly a 6000-fold increase in viral titers during the 48 h infection period. However, mhTERTAd.sTbetaRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication-deficient virus Ad(E1(-)).sTbetaRFc also produced high levels of sTGFbetaRIIFc, but under these conditions, no detectable viral replication was observed. Adenoviral-mediated production of sTGFbetaRIIFc was shown to bind with TGFbeta-1, and to abolish the effects of TGFbeta-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTbetaRFc intravenously into MDA-MB-231 human xenograft-bearing mice resulted in a significant inhibition of tumor growth and production of sTGFbetaRIIFc in the blood. Conversely, intravenous injection of Ad(E1(-)).sTbetaRFc did not show a significant inhibition of tumor growth, but resulted in sTGFbetaRIIFc in the blood, suggesting that viral replication along with s

  14. A modified hTERT Promoter-directed Oncolytic Adenovirus Replication with Concurrent Inhibition of TGFβ Signaling for Breast Cancer Therapy

    Science.gov (United States)

    Hu, Zebin; Robbins, John S.; Pister, Amanda; Zafar, M. Behzad; Zhang, Zhen-Wei; Gupta, Janhavi; Lee, K. Jessica; Neuman, Kam; Yun, Chae-Ok; Guise, Theresa; Seth, Prem

    2009-01-01

    Our laboratory is interested to develop oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we have constructed mhTERTAd.sTβRFc, a 01/07 based adenoviral vector expressing the soluble form of TGFβ receptor II fused with human Fc IgG1 (sTGFβRIIFc) gene, in which viral replication is under the control of modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTβRFc-mediated sTGFβRIIFc production would target growth factor-β (TGFβ) pathway known to contribute to the tumor progression breast cancer metastasis. We chose to use mhTERT promoter because it was found to be relatively more active (approximately 20-times) in breast cancer cells compared to normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 hrs with mhTERTAd.sTβRFc produced high levels of sTGFβRIIFc (greater than 1 μg/ml) in the medium. Breast cancer cells produced nearly 6,000-fold increase in the viral titers during 48 hrs infection period. However, mhTERTAd.sTβRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication deficient virus Ad(E1-).sTβRFc also produced high levels of sTGFβRIIFc, but under these conditions no detectable viral replication was observed. Adenoviral-mediated production of sTGFβRIIFc was shown to bind with TGFβ-1, and abolished the effects of TGFβ-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTβRFc intravenously into MDA-MB-231 human xenograft bearing mice resulted in significant inhibition of tumor growth, and production of sTGFβRIIFc in the blood. On the other hand, intravenous injection of Ad(E1-).sTβRFc did not exhibit significant inhibition of the tumor growth, but resulted in the sTGFβRIIFc in the blood, suggesting that viral replication along with sTGFβRIIFc protein production play a critical role in inducing

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

    Science.gov (United States)

    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

  16. 条件复制型腺病毒的研究进展%Advance of research in conditionally replicating adenovirus

    Institute of Scientific and Technical Information of China (English)

    邬朝平

    2011-01-01

    条件复制型腺病毒(conditionally replicating adenovirus,CRAd)基因治疗肿瘤的研究主要集中于提高其靶向性、增强肿瘤杀伤特异性,以及降低毒性作用三个方面.在提高CRAd靶向性的研究中,早期策略主要集中在腺病毒早期复制必需基因Ela/Elb突变或缺失的调控以及肿瘤或组织特异性启动子对其转录的调控;近年来利用组织特异性microRNA对病毒早期复制必需基因的转录后调控以及病毒外壳蛋白修饰的转导调控已逐渐成为研究的热点.在提高CRAd的肿瘤杀伤方面,除删除病毒自身凋亡抑制基因与导入外源性治疗基因两种方法以外,改造外壳纤毛提高病毒对靶细胞的亲嗜性及构建靶向肿瘤干细胞的CRAd成为新的关注点.同时,随着对腺病毒结构认识的逐步加深,修饰其外壳六邻体蛋白及其他外壳蛋白以降低CRAd的肝嗜性与免疫原性也正成为降低CRAd毒性作用的研究热点.

  17. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody

    National Research Council Canada - National Science Library

    Sainsbury, Frank; Sack, Markus; Stadlmann, Johannes; Quendler, Heribert; Fischer, Rainer; Lomonossoff, George P

    2010-01-01

    .... To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non...

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

    Science.gov (United States)

    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.

  19. Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage.

    Science.gov (United States)

    Wong, S P; Argyros, O; Coutelle, C; Harbottle, R P

    2011-01-01

    The ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.

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

    Directory of Open Access Journals (Sweden)

    Daniel M Appledorn

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

  1. [Influence of Japanese enciphalitis virus capsid protein on the self-replicate ability of JEV replicon vectors].

    Science.gov (United States)

    Huang, Ying; Liu, Shan; Yang, Peng; Wang, Chao; Du, Yun; Sun, Zhiwei; Yu, Weiyuan

    2010-08-01

    To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.

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

    Science.gov (United States)

    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.

  3. Oncolytic Adenoviruses in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Ramon Alemany

    2014-02-01

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

  4. Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

    Science.gov (United States)

    Farley, Daniel C; Bannister, Richard; Leroux-Carlucci, Marie A; Evans, Nerys E; Miskin, James E; Mitrophanous, Kyriacos A

    2012-10-01

    The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.

  5. The Early Activation of CD8+ T Cells Is Dependent on Type I IFN Signaling following Intramuscular Vaccination of Adenovirus Vector

    Directory of Open Access Journals (Sweden)

    Masahisa Hemmi

    2014-01-01

    Full Text Available Few of the vaccines in current use can induce antigen- (Ag- specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m. vaccination of an adenovirus vector (Adv can induce Ag-specific cytotoxic T lymphocytes (CTLs in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs, which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.

  6. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available The highly pathogenic avian influenza (HPAI H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138, and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine.

  7. Suppressing tumor growth of nasopharyngeal carcinoma by hTERTC27 polypeptide delivered through adeno-associated virus plus adenovirus vector cocktail

    Institute of Scientific and Technical Information of China (English)

    Xiong Liu; Xiang-Ping Li; Ying Peng; Samuel S.Ng; Hong Yao; Zi-Feng Wang; Xiao-Mei Wang; Hsiang-Fu Kung; Marie C.M.Lin

    2012-01-01

    Nasopharyngeal carcinoma(NPC) is a metastatic carcinoma that is highly prevalent in Southeast Asia.Our laboratory has previously demonstrated that the C-terminal 27-kDa polypeptide of human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human glioblastoma and melanoma cells.In this study,we investigated the antitumor effect of hTERTC27 in human C666-1 NPC cells xenografted in a nude mouse model.A cocktail of vectors comprising recombinant adeno-associated virus (rAAV) and recombinant adenovirus (rAdv) that each carry hTERTC27 (rAAV-hTERTC27 and rAdv-hTERTC27; the cocktail was abbreviated to rAAV/rAdv-hTERTC27) was more effective than either rAAV-hTERTC27 or rAdv-hTERTC27 alone in inhibiting the growth of C666-1 NPC xenografts.Furthermore,we established three tumors on each mouse and injected rAAV/rAdv-hTERTC27 into one tumor per mouse.Although hTERTC27 expression could only be detected in the injected tumors,reduced tumor growth was observed in the injected tumor as well as the uninjected tumors,demonstrating that the vector cocktail could provoke an antitumor effect on distant,metastasized tumors.Further studies showed the observed antitumor effects included inducing necrosis and apoptosis and reducing microvessel density.Together,our data suggest that the rAAV/rAdv-hTERTC27 cocktail can potently inhibit NPC tumor growth in both local and metastasized tumors and should be further developed as a novel gene therapy strategy for NPC.

  8. The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: Implications for the mechanism of DNA replication

    NARCIS (Netherlands)

    Steenbergh, P.H.; Sussenbach, J.S.

    The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the

  9. The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: Implications for the mechanism of DNA replication

    NARCIS (Netherlands)

    Steenbergh, P.H.; Sussenbach, J.S.

    1979-01-01

    The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the

  10. Protection of Nonhuman Primates Against Two Species of Ebola Virus Infection With a Single Complex Adenovirus Vector

    Science.gov (United States)

    2010-04-01

    Zaire Ebola virus . Virology 346:394–401. 21. Leffel, E. K., and D. S. Reed. 2004. Marburg and Ebola viruses as aerosol threats. Biosecur. Bioterror. 2...the event of a natural virus outbreak or biological threat. The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe...vaccine efficacy are justifiable. While the combination of the two CAdVax-based vectors for Ebola virus (EBO7) and Marburg virus (M8) GPs constitute the

  11. Development of a novel adenovirus-alphavirus hybrid vector with RNA replicon features for malignant hematopoietic cell transduction.

    Science.gov (United States)

    Yang, Y; Xiao, F; Lu, Z; Li, Z; Zuo, H; Zhang, Q; Li, Q; Wang, H; Wang, L-S

    2013-08-01

    To improve the expression levels of transgenes in malignant hematopoietic cells, we developed a novel adenoviral-alphavirus hybrid vector Ad5/F11p-SFV-GFP that contains a Semliki Forest Virus (SFV) replicon and chimeric fibers of Ad5 and Ad11p. Ad5/F11p-SFV-GFP infected >95% of K562, U937 or Jurkat cells and 23.65% of HL-60 cells, and led to moderate Enhanced Green Fluorescent Protein (EGFP) transgene expression intensity. The infection efficiency of Ad5/F11p-SFV-GFP in primary human leukemia cells ranged from 9.34-89.63% (median, 28.58%) at a multiplicity of infection (MOI) of 100, compared with only 3.37-44.54% (median, 10.42%) in cells infected by Ad5/F11p-GFP. Importantly, Ad5/F11p-SFV-GFP led to a significantly higher transgene expression level in primary leukemia cells, as indicated by the relative fluorescence intensity, compared to cells infected with Ad5/F11p-GFP. The increased expression of EGFP in Ad5/F11p-SFV-GFP-infected cells was associated with the accumulation of abundant subgenomic mRNA. Additionally, infection of K562, U937 or Jurkat cells by Ad5/F11p-SFV-GFP was significantly inhibited by blocking CD46 receptor; however, other factors may affect the gene-transfer efficiency of Ad5/F11p-SFV-GFP in primary leukemia cells. In conclusion, we successfully developed a novel adenoviral-alphavirus hybrid vector with RNA replicon features, which represents a promising vector for gene modifications during the production of cell-based vaccines for leukemia patients.

  12. Development of conditionally replicating integrase defective lentiviral vectors for Epstein-Barr virus gene therapy

    OpenAIRE

    Blasi, Maria

    2012-01-01

    Integrase defective lentiviral vectors show promise for achieving gene expression without integration, preserving some benefits of LVs, whereas reducing the potentially detrimental risk of insertional mutagenesis. Numerous reports supported the ability of these vectors to confer long-term gene expression in slowly dividing cell types for potentially corrective gene expression. These reports also highlighted additional applications of these vectors as delivery platforms for alternate integrati...

  13. Co-expression of Erns and E2 genes of classical swine fever virus by replication-defective recombinant adenovirus completely protects pigs against virulent challenge with classical swine fever virus.

    Science.gov (United States)

    Sun, Yongke; Yang, Yuai; Zheng, Huanli; Xi, Dongmei; Lin, Mingxing; Zhang, Xiaomin; Yang, Linfu; Yan, Yulin; Chu, Xiaohui; Bi, Baoliang

    2013-04-01

    The objective of this study was to construct a recombinant adenovirus for future CSFV vaccines used in the pig industry for the reduction of losses involved in CSF outbreaks. The Erns and E2 genes of classical swine fever virus (CSFV), which encode the two main protective glycoproteins from the "Shimen" strain of CSFV, were combined and inserted into the replication-defective human adenovirus type-5 and named the rAd-Erns-E2. Nine pigs were randomly assigned to three treatment groups (three pigs in each group) including the rAd-Erns-E2, hAd-CMV control and DMEM control. Intramuscular vaccination with 2×10(6) TCID(50) of the rAd-Erns-E2 was administered two times with an interval of 21 days. At 42 days post inoculation, pigs in all groups were challenged with a lethal dose of 1×10(3) TCID(50) CSFV "Shimen" strain. Observation of clinical signs was made and the existence of CSFV RNA was detected. Animals in the hAd-CMV and DMEM groups showed severe clinical CSF symptoms and were euthanized from 7 to 10 days after the challenge. However, no adverse clinical CSF signs were observed in vaccinated pigs after the administration of rAd-Erns-E2 and even after CSFV challenge. Neither CSFV RNA nor pathological changes were detected in the tissues of interest of the above vaccinated pigs. These results implied that the recombination adenovirus carrying the Erns-E2 genes could be used to prevent swine from classical swine fever.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    - either by inducing cross-reactive T cells or by administering a polyvalent vaccine. To test these strategies, we designed 3 ancestral and 2 circulating sequences based on the two domains of the E1 and E2 proteins of papillomaviruses (PVs) that exhibit the highest degree of conservation in comparison...... to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...... circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did...

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

    Science.gov (United States)

    WU, JIE; CHEN, KE-DA; GAO, MENG; CHEN, GANG; JIN, SU-FENG; ZHUANG, FANG-CHENG; WU, XIAO-HONG; JIANG, YUN-SHUI; LI, JIAN-BO

    2015-01-01

    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 6.31×109 IU/ml and 3.0×109 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 109 IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ2MSB=20.00 and χ2WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development. PMID:25780403

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

    Science.gov (United States)

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

    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.

  17. 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.

    Science.gov (United States)

    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.

  18. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.

    Science.gov (United States)

    Gong, Shiaoching; Yang, Xiangdong William; Li, Chenjian; Heintz, Nathaniel

    2002-12-01

    Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

  19. Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer.

    OpenAIRE

    Pulsinelli, G A; Temin, H M

    1991-01-01

    Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing ...

  20. Construction and expression of recombinant adenovirus vector encoding BDNF%重组大鼠脑源性神经营养因子腺病毒的构建及体外表达分析

    Institute of Scientific and Technical Information of China (English)

    郎洪刚; 曹文斌; 牛道立; 何芬

    2011-01-01

    Objective To construct adenovirus vector encoding rat brain derived neurotrophic factor (BDNF) gene and identify the expression in vitro.Methods The specific BDNF sequence was cloned into the plasmid of pAdTrack-CMV to construct the BDNF expression plasmid pAd-BDNF.The recombinant plasmids were identified hy DNA sequencing and restriction digestion.The homologous recomhination between the recombinant vector and the adenovirus hone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-BDNF.The infecting recomhinant virus particles Ad-BDNF was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.The recombinant adenovirus vector infected the hela cell, the expression of BDNF was detected by RT-PCR, Western blot and immunocytochemistry.Results The sequence results of pAd-BDNF were consistent with expectancy.After homologous recombination and packaging with 293 cells , the recombinant Ad-BDNF adenovirus was obtained.RT-PCR, Western blot and immunocytochemistry suggested that the BDNF was expressed.Conclusions The Ad-BDNF was constructed successfully.It is confirmed that the interest proteins were expressed in the infected cells , which lays the basis for its application in the treatment of the neurodamaged diseases.%目的 构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况.方法 将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-BDNF).重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况.结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒

  1. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    OpenAIRE

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing

    2013-01-01

    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 effica...

  2. 重组干扰素慢病毒共感染对腺病毒体外复制的影响%The effect of recombinant interferon lentivirus co-infection on adenovirus in vitro replication

    Institute of Scientific and Technical Information of China (English)

    何小艳; 翁云层; 张艺凡; 张云; 帅丽芳; 李红卫; 黎诚耀; 张玲

    2015-01-01

    目的:测定不同启动子的重组干扰素(IFN)慢病毒(LV)共感染对5型腺病毒在体外293A细胞中复制的影响。方法以重组LV-EF1α-IFNγ、LV-CMV-IFNα、LV-CMV-IFNγ、LV-Ubi-IFNα与5型腺病毒共感染293A细胞,72 h后,荧光定量PCR法测定5型腺病毒载量。结果 LV-EF1α-IFNγ、LV-CMV-IFNα、LV-CMV-IFNγ、LV-Ubi-IFNα共感染组的腺病毒载量低于非共感染对照组( P <0.01)。结论使用重组LV-IFN感染293A后,能够显著抑制腺病毒复制,为进一步开展HIV/AIDS等病毒性疾病的基因治疗研究提供参考。%Objective To determine the inhibition effect of recombinant interferon lentivirus co-infection with adenovirus 5 a-mong 293A cells in vitro .Methods Recombinant LV-EF1α-IFNγ, LV-CMV-IFNα,LV-CMV-IFNγ, LV-Ubi-IFNαand adenovirus 5 co-infection among 293A cells were used and 72 hours later, fluorescence quantitative PCR method was adopted to detect adenovirus 5 viral loads.Resulst Viral loads of LV-EF1α-IFNγ, LV-CMV-IFNα, LV-CMV-IFNγ, LV-Ubi-IFNαco-infection group were signifi-cantly lower than those of control group( P <0.01).Conclusion LV-IFN co-infection can inhibit adenovirus replication among 293A cells, which lays the foundation for the gene therapy of viral diseases such as HIV/AIDS.

  3. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

    Science.gov (United States)

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J Michael; Kanerva, Anna; Hemminki, Akseli

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors.

  4. Monitoring of Biodistribution and Persistence of Conditionally Replicative Adenovirus in a Murine Model of Ovarian Cancer Using Capsid-Incorporated mCherry and Expression of Human Somatostatin Receptor Subtype 2 Gene

    Directory of Open Access Journals (Sweden)

    Igor P. Dmitriev

    2014-10-01

    Full Text Available A significant limiting factor to the human clinical application of conditionally replicative adenovirus (CRAd-based virotherapy is the inability to noninvasively monitor these agents and their potential persistence. To address this issue, we proposed a novel imaging approach that combines transient expression of the human somatostatin receptor (SSTR subtype 2 reporter gene with genetic labeling of the viral capsid with mCherry fluorescent protein. To test this dual modality system, we constructed the Ad5/3Δ24pIXcherry/SSTR CRAd and validated its capacity to generate fluorescent and nuclear signals in vitro and following intratumoral injection. Analysis of 64Cu-CB-TE2A-Y3-TATE biodistribution in mice revealed reduced uptake in tumors injected with the imaging CRAd relative to the replication-incompetent, Ad-expressing SSTR2 but significantly greater uptake compared to the negative CRAd control. Optical imaging demonstrated relative correlation of fluorescent signal with virus replication as determined by viral genome quantification in tumors. Positron emission tomography/computed tomography studies demonstrated that we can visualize radioactive uptake in tumors injected with imaging CRAd and the trend for greater uptake by standardized uptake value analysis compared to control CRAd. In the aggregate, the plasticity of our dual imaging approach should provide the technical basis for monitoring CRAd biodistribution and persistence in preclinical studies while offering potential utility for a range of clinical applications.

  5. Therapeutic induction of angiogenesis by direct myocardial administration of an adenovirus vector encoding human hepatocyte growth factor gene and its safety

    Institute of Scientific and Technical Information of China (English)

    WU Danli; ZHANG Yourong; LAO Miaofen; YUAN Lizhen; WANG Lan; HA Xiaoqin; WU Zuze(WU Cutse)

    2004-01-01

    After the study in vitro and in rats, we assessed further the effects and safety of local angiogen therapy using intramyocardial delivery of an adenovirus carrying hepatocyte growth factor gene (Ad-HGF) in a canine ischemia model. The angiogenic activity of Ad-HGF was evaluated from three aspects. First, the augmentation of collateral vessel development was assessed by angiography 30 d after surgery. The results showed that the density of collateral vessels in treated group was higher than that of control group. Secondly, infarct size was evaluated by TTC staining and image analysis. The results showed that the infarct size of treated group was smaller than that of control group. Thirdly, the myocardial regional blood flow was determined by the method of colored microspheres. The results showed that the blood flow recovered to the level before ligation in treated group, but that of the control group was lower than normal level. In addition, during the study of chronic toxicity, we tested the anti-adenovirus antibodies by neutralization method. The antibodies yielded after the fourth injection decreased slowly from peak level and disappeared 12 weeks after drug withdrawal. Overall, Ad-HGF can promote angiogenesis in ischemic myocardium and reduce infarct size.So this method may be considered as a therapeutic angiogenesis induction strategy for ischemic disease including myocardial infarction and peripheral artery disease. At the same time, Ad-HGF could induce the yield of anti-adenovirus antibodies to neutralize adenovirus, which may be the mechanism of adenovirus clearance.

  6. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L

    1999-01-01

    We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation o...

  7. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    Science.gov (United States)

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G

    1998-06-12

    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  8. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WEI Xue-lei; LIN Lin; HOU Yu; FU Xin; ZHANG Ji-ying; MAO Ze-bin; YU Chang-long

    2008-01-01

    Background Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-1RES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-1RES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR.Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβI and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen typeⅢ, and fibronectin mRNA among matrix markers.Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

  9. Vectores

    OpenAIRE

    2016-01-01

    Documento que contiene la explicación sobre las temáticas de Sistemas coordenados, Cantidades vectoriales y escalares, Algunas propiedades de los vectores, Componentes de un vector y vectores unitarios

  10. Construction of recombinant adenovirus vector containing AFP and generation of adenovirus-mediated AFP gene modified dendritic cells vaccine%含人AFP基因重组腺病毒载体的构建及其转染树突状细胞瘤苗的制备

    Institute of Scientific and Technical Information of China (English)

    杨静悦; 曹大勇; 刘文超; 斯小明

    2009-01-01

    Objective:To construct recombinant adenovirus vectors containing human AFP genes,and infect dendritic cell. Methods: Full length AFP cDNAs were subcloned into pIND vector,followed by being cloned into shuttle2 vector.The AFP gene fragments resulted from the shuttle2-AFP digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA.After packaged with HEK293 cells,the adenovirus expression vector was obtained.The plasmid pAdeno-AFP was identified by endonuclease and PCR.After dendritic cells were infected pAdeno-AFP,the surface molecules of pAdeno-AFP/DC were analysed by flow cytometry.AFP levels in culture supernatant of pAdeno-AFP/DC were measured by ELISA. Results: AFP gene in the inserted DNA of adeno-AFP was confirmed by PCR,and predictive fragments proved by restriction enzyme digestion analysis were exhibited.All the above results indicated that human AFP gene had been connected with pAdeno-X vectors correctly.The recombinant adenovirus vector of human AFP gene packaged in HEK293 cells,it will be used to introduce the target gene into dendritic cell.pAdeno-AFP/DC were able to upregulate CD1a,CD11c,CD80,CD86 and HLA-DR.And pAdeno-AFP/DC could secrete high level of AFP in vitro. Conclusion: The recombinant adenovirus vector of human AFP gene have been constructed successfully.The established AFP -DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy,and it will be the foundation of future clinical use of DC vaccine.%目的:构建含人AFP基因的腺病毒载体,体外转染树突状细胞,制备树突状细胞肝癌瘤苗.方法: 将AFP基因亚克隆到pIND 载体和Shuttle2载体中,构建穿梭载体Shuttle2-AFP.用PI-Sce Ⅰ和I-CeuⅠ双酶切后将所获AFP基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-AFP重组腺病毒载体.其后,用重组腺病毒载体转染HEK293细胞,包装腺病毒表达载体.通过酶切、PCR对腺病毒载体进行鉴定.包装好的重组病毒载体pAdeno-AFP体外

  11. 羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

    Institute of Scientific and Technical Information of China (English)

    邵洪泽; 毛文智; 宋阳; 李琳; 程荣华; 孙健; 孙强

    2010-01-01

    [目的]利用腺病毒载体系统构建羊传染性脓疱病毒B2L基因重组腺病毒载体.[方法]以从羊传染性脓疱病毒株JLSY04中提取的基因组DNA为模板,PCR扩增获得B2L目的基因片段;然后将B2L目的基因克隆至PDNR-CMV载体,筛选阳性克隆获得质粒CTC572-6;再将质粒CTC572-6与腺病毒载体进行同源重组,筛选阳性克隆,并进行菌液PCR、酶切、测序等鉴定.[结果]经酶切和基因测序等鉴定,成功构建了携带羊传染性脓疱病毒B2L基因的重组腺病毒载体CTC572Ade-30.[结论]为羊传染性脓疱基因工程疫苗的进一步研究奠定基础.%[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification; B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained; CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

  12. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy.

    Science.gov (United States)

    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.

  13. Roles for the E4 orf6, orf3, and E1B 55-kilodalton proteins in cell cycle-independent adenovirus replication.

    Science.gov (United States)

    Goodrum, F D; Ornelles, D A

    1999-09-01

    Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.

  14. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  15. A Tetravalent Dengue Vaccine Based on a Complex Adenovirus Vector Provides Significant Protection in Rhesus Monkeys against All Four Serotypes of Dengue Virus▿

    OpenAIRE

    Raviprakash, Kanakatte; Wang, Danher; Ewing, Dan; Holman, David H.; Block, Karla; Woraratanadharm, Jan; Chen, Lan; Hayes, Curtis; Dong, John Y.; Porter, Kevin

    2008-01-01

    Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vect...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-10-01

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

  17. Characterization of Infectivity-Enhanced Conditionally Replicating Adenovectors for Prostate Cancer Radiovirotherapy

    OpenAIRE

    Oneal, Michael J.; Trujillo, Miguel A; Davydova, Julia; McDonough, Samantha; Yamamoto, Masato; Morris, John C.

    2012-01-01

    Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with r...

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

    Directory of Open Access Journals (Sweden)

    Chitose Kurihara

    2016-12-01

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

  19. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    Institute of Scientific and Technical Information of China (English)

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍

    2003-01-01

    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  20. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  1. Adenovirus-mediated Gene Transfer of MMP-2 into Cultured Porcine Trabecular Meshwork Cells

    OpenAIRE

    2012-01-01

    This study aimed to use adenoviral gene transfer to express matrix metalloproteinase (MMP)-2 in cultured porcine trabecular meshwork cells and to evaluate the duration of adenovirus-mediated MMP-2 expression and its enzymatic activity. MMP-2 cDNA was synthesized by ligating three segments of MMP-2 cDNA obtained by reverse transcription-polymerase chain reaction (RT-PCR) with mRNA extracted from mouse lungs. MMP-2 cDNA was inserted into replication-deficient adenoviral vectors. Western blottin...

  2. Adenovirus-mediated nitric oxide synthase gene transfer.

    Science.gov (United States)

    Raman, Kathleen G; Shapiro, Richard A; Tzeng, Edith; Kibbe, Melina R

    2004-01-01

    The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

  3. Construction of recombinant adenovirus vector of mouse keratinocyte growth factor receptor%小鼠角质化生长因子受体(KGFR)重组腺病毒载体的构建

    Institute of Scientific and Technical Information of China (English)

    陈力; 曹春蕾; 李蓓; 周璇; 李银平; 黎檀实

    2011-01-01

    Objective To construct a recombinant adenovirus vector expressing keratinocyte growth factor receptor (KGFR) gene of mouse. Methods KGFR gene was amplified by polymerase chain reaction (PCR) from mouse cDNA which had been reverse transcripted from RNA harvested from C57BL/6 mouse, and was then inserted into the plasmid pShuttle-CMV to construct the shuttle plasmid pShuttle-CMV-KGFR (pKGFR). After linearized by restriction enzyme, the plasmid was transformed into Escherichia. coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR) was identified, linearized, and then transfected into HEK293 cells using lipofectamineTM 2000 to package the adenovirus, Adeasy-1-pShuttle-CMV-KGFR (Ad-KGFR), followed by further amplification, caesium chloride density gradient centrifugation purification and measurement of virus titer. Results Firstly, the shuttle plasmid pKGFR was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. Then, the cytopathic effects of HEK293 cells ohserved under the microscope suggested the successful duplication of the virus. Finally, the plague titration of on HEK293 cells showed virus titers were 2.25 × 1010 PFU/ml, which was enough concentration for future test in vivo or in vitro.Conclusion The recombinant adenovirus vector of pAd-KGFR is constructed successfully to be used in the future test to investigate the effects of KGFR in pulmonary diseases, and provides attractive therapeutic target in the pulmonary disease.%目的 构建表达小鼠KGFR基因的重组腺病毒.方法 提取小鼠肺组织RNA,RT-PCR获得cDNA后,PCR扩增目的 基因片段.将获得的KGFR基因插入载体质粒pShuttle-CMV,构建穿梭质粒pShuttle-CMV-KGFR(pKGFR).线性化pKGFR后,转化入含Adeasy-1病毒骨架的BJ5183细菌中.筛选正确的同源重组质粒pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR),转染HEK293细胞,产生病毒颗粒Adeasy-1

  4. Biobased monoliths for adenovirus purification.

    Science.gov (United States)

    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.

  5. 小鼠CD200基因重组腺病毒载体的构建及鉴定%Construction and identification of recombinant adenovirus vector containing mouse CD200 gene

    Institute of Scientific and Technical Information of China (English)

    王琼; 陈冬梅; 王立斌; 魏军; 李玉奎

    2013-01-01

    Objective:To construct the recombinant adenovirus vector containing mouse CD200 gene and to detect its expression in mice pancreas endothelial cells(mile sven 1,MS1).Methods:mCD200 gene was subcloned into the shuttle plasmid pYr-adshuttle-4 and recombinant plasmid pYr-ads-4-mCD200 was acquired.After that mCD200 mRNA expression frame was transfered to pAd/PLDEST adenovirus vector via LR(attL × attR) in vitro homologous recombination from pYr-ads-4-mCD200.Recombinant adenovirus plasmid was digested by Pac Ⅰ and transfected into HEK 293 cells and packaged out recombinant adenovirus rAd-4-mCD200,which was identified by digestion and PCR analysis.Virus titer was measured by the tissue cell infectious dosage 50(TCID50) method.Meanwhile,adenovirus was amplified in HEK 293 cells and used to infect MS1 cells.Expression of mCD200 gene was measured by fluorescence microscope and real-time PCR.Results:PCR identification demonstrated the construction of recombinant adenovirus carrying rAd-4-mCD200 was successful and its titer was about 109-1010 pfu/ml after amplification.Through the fluorescence microscope and real-time PCR,recombinant adenoviral vector of mCD200 gene was constructed successfully in the MS1 cells.Conclusions:MS1 cells infected by constructed adenovirus vector rAd-4-mCD200 could express mCD200 successfully and may provide the foundation for the next step to carry out the CD200 gene immunosuppression regulation and other related study.%目的:构建携带小鼠CD200基因的重组腺病毒载体,并鉴定其在小鼠胰腺内皮细胞(mile sven 1,MS1)中mCD200基因的表达情况.方法:mCD200基因亚克隆至腺病毒穿梭质粒pYr-adshuttle-4,获得重组质粒pYr-ads-4-mCD200.从该重组质粒上利用LR(attL×attR)特异性同源重组的方法,将mCD200表达框构建至腺病毒骨架质粒pAd/PL-DEST上,获得的重组腺病毒质粒经PacⅠ酶切,转染HEK293细胞,包装成重组腺病毒rAd-4-mCD200,并用酶切和PCR等鉴定.采用TCID 50

  6. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    OpenAIRE

    Sun X; Zhang Z; Gong T; Zhao D.; Han J; Zeng Q

    2012-01-01

    Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, w...

  7. Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types.

    Science.gov (United States)

    Twitty, Chris G; Diago, Oscar R; Hogan, Daniel J; Burrascano, Cindy; Ibanez, Carlos E; Jolly, Douglas J; Ostertag, Derek

    2016-02-01

    Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6 μg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals.

  8. Construction of adenovirus vector expressing SDF-1/RUNX1 and detection of viral titre%SDF-1/RUNX1融合蛋白腺病毒载体的构建及滴度测定

    Institute of Scientific and Technical Information of China (English)

    罗红春; 张红宾; 秦波; 汪嘉莉; 刘倩

    2012-01-01

    目的 构建SDF-1/RUNX1融合蛋白腺病毒表达载体,并测定其滴度,为研究SDF-1/RUNX1融合蛋白介导的间充质干细胞对造血干细胞定向募集的作用打下基础.方法 全基因合成SDF-1-(GlySer)3-RUNX1片段,并在其两端添加限制性酶切位点XhoI及EcoRI,经测序验证基因序列是否正确.采用分子克隆技术构建pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体,转染入293A细胞中进行包装,采用免疫法测定腺病毒滴度.结果 全基因合成SDF-1-(GlySer)3-RUNX1片段经测序验证基因序列正确,长约3 000 bp.将其连接至目的 载体pIRES2-EGFP,构建出含有SDF-1-(GlySer)3-RUNX1基因序列的GFP共表达质粒编号为B.以B质粒为模板,扩增出带attB1和attB2位点的SDF-1-(GlySer)3-RUNX1-IRES-EGFP片段,长约4 300 bp.采用BP重组系统将上述目的 片段重组到载体pDONR221,构建出BP重组质粒C.再采用LR重组系统将目的 序列重组到腺病毒载体pAD/CMV/v5-DEST上,构建出pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体.经293细胞包装后,采用免疫法测定腺病毒滴度为1.03×1011 ifu/mL.结论 成功构建出SDF-11/RUNX1融合蛋白腺病毒表达载体,且腺病毒滴度较高,有利于后续试验.%To construct SDF1/RUNX1 fusion protein adenovirus vector, and to assay its titre, in order to lay the groundwork for researching the oriented recruitment of hematopoietic stem cells influenced by mesenchymal stem cells mediated by SDF1/RUNX1 fusion protein. Methods We synthesized SDF-l-(GlySer) 3-RUNX1 gene fragment with restriction enzyme cutting sites of Xhol and EcoRI at both ends, and sequenced the SDF-l-(GlySer) 3-RUNX1 gene fragment. We constructed pAD-SDF-1-(GlySer)3-RUNX1-IRKS-GFP adenovirus vector by molecular cloning,transfected this adenovirus vector into 293A cell strain for packing,and assayed virus titre using immunization. Results We synthesized SDF-l-(GlySer)3-RUNXl gene fragment successfully,and it was about 3000bp. This right gene

  9. 利用腺病毒载体介导的RNA干扰技术在悬浮细胞中获得基因沉默效应%Adenovirus expression vector mediated gene silencing in suspension cells

    Institute of Scientific and Technical Information of China (English)

    王淳; 方文刚; 李波

    2012-01-01

    目的 利用重组腺病毒载体携带短发夹RNA (shRNA),使难于转染的悬浮细胞达到较高的目的基因沉默效率.方法 分别将含有人胎盘生长因子(PLGF)的干扰序列及阴性对照序列(scramble)连入pRNAT-H1.1/Adeno载体,获得重组穿梭质粒;将线性化的重组穿梭质粒与腺病毒骨架重组;将重组子转染HEK 293细胞,经过3轮病毒包装,收获病毒颗粒并测定滴度.病毒颗粒感染悬浮细胞——人小细胞肺癌细胞(NCI-H 250和NCI-H 209),通过实时定量PCR方法鉴定靶基因沉默效应,并利用肿瘤细胞重建基质膜侵袭实验进行功能鉴定.结果 成功构建带有靶基因干扰序列及scramble、空载体的3种腺病毒重组子(AD-shuttle-shPLGF、AD-shuttlescramble和AD-shuttle).经过HEK 293细胞包装,最终获得3株病毒颗粒,病毒滴度分别为1.5×1011、1.6×1011和1.6×1011.NCI-H 250和NCI-H 209细胞感染腺病毒颗粒48 h后,大部分细胞表达GFP,感染率接近100%;且两株细胞感染AD-shuttle-shPLGF病毒48 h后,PLGF mRNA表达水平降低(P<0.01),肿瘤细胞侵袭能力明显下降(P<0.01).结论 携带shRNA的腺病毒表达载体可以代替电转、脂质体介导等方法,使难于转染细胞的目的基因达到有效沉默.%Objective Using recombinant adenovirus to make gene silencing in suspension cell. Methods Placen-tal growth factor (PLGF) silencing target sequence and scramble sequence were inserted into shuttle plasmid pR-NAT-H1. 1/Adenb respectively to gain recombinant shuttle vectors. Then the two recombinant shuttle vectors and empty vector were co-transformed with pAdeasy-1 through homologous recombination. These recombinant adenovi-ruses were packaged and amplified in HEK 293 cells. Human small cell lung cancer cells NCI-H 250 and NCI-H 209, were infected by recombinant adenovirus. And the expression of target gene was detected by real - time PCRand cancer cells' invation function was analyzed by Transwell cancer cell

  10. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    Science.gov (United States)

    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. Enhanced structural stability of adenovirus nanocapsule

    Institute of Scientific and Technical Information of China (English)

    Ding Weng; Ziyue Karen Jiang; Jing Jin; Lily Wu; Yunfeng Lu

    2014-01-01

    Application of viral vector in gene therapy and vaccination is still limited by their structural stability, which significantly increased avoidable cost in storage and transportation. Herein a non-covalent conjugated low-pH degradable nanocapsule has been adopted to stabilize viral vectors. By utilizing a luciferase expressing adenovirus, AdCMVLuc, we succeeded in a raise of over 11 folds in AdCMVLuc's structural stability after 12 days storage at 4 1C.

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

    Science.gov (United States)

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

    2012-10-01

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

  13. A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer

    NARCIS (Netherlands)

    Riezebos-Brilman, A.; Walczak, M.; Regts, J.; Rots, Mg; Kamps, G.; Dontje, B.; Haisma, Hy; Wilschut, J.; Daemen, T.

    2007-01-01

    Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical

  14. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    Science.gov (United States)

    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.

  15. Predicted structure of two adenovirus tumor antigens.

    OpenAIRE

    Perricaudet, M; Le Moullec, J M; Pettersson, U

    1980-01-01

    Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C...

  16. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

    DEFF Research Database (Denmark)

    Bahrami, Shervin; Pedersen, Finn Skou; Duch, Mogens R.

    2003-01-01

    envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random......The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV...... mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells...

  17. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis

    Directory of Open Access Journals (Sweden)

    Andreas Jacobs

    1999-11-01

    Full Text Available The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1 virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.

  18. First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens.

    Science.gov (United States)

    Nyombayire, Julien; Anzala, Omu; Gazzard, Brian; Karita, Etienne; Bergin, Philip; Hayes, Peter; Kopycinski, Jakub; Omosa-Manyonyi, Gloria; Jackson, Akil; Bizimana, Jean; Farah, Bashir; Sayeed, Eddy; Parks, Christopher L; Inoue, Makoto; Hironaka, Takashi; Hara, Hiroto; Shu, Tsugumine; Matano, Tetsuro; Dally, Len; Barin, Burc; Park, Harriet; Gilmour, Jill; Lombardo, Angela; Excler, Jean-Louis; Fast, Patricia; Laufer, Dagna S; Cox, Josephine H

    2017-01-01

     We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)-vectored, human immunodeficiency virus type 1 (HIV-1) vaccine.  Sixty-five HIV-1-uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35-vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH).  All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot-determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8(+) T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic.  SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime

  19. Inhibition of hepatitis B virus replication by helper dependent adenoviral vectors expressing artificial anti-HBV pri-miRs from a liver-specific promoter.

    Science.gov (United States)

    Mowa, Mohube Betty; Crowther, Carol; Ely, Abdullah; Arbuthnot, Patrick

    2014-01-01

    Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

  20. Inhibition of Hepatitis B Virus Replication by Helper Dependent Adenoviral Vectors Expressing Artificial Anti-HBV Pri-miRs from a Liver-Specific Promoter

    Directory of Open Access Journals (Sweden)

    Mohube Betty Mowa

    2014-01-01

    Full Text Available Research on applying RNA interference (RNAi to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR promoter for expression of anti-HBV primary microRNAs (pri-miRs. HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

  1. Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2011-06-01

    Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

  2. Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus.

    Directory of Open Access Journals (Sweden)

    Daehwan Chung

    Full Text Available The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105, and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

  3. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

  4. Construction and immunogenicity of Aβ42 and GM-CSF coexpression adenovirus vector%Aβ42及GM-CSF双基因重组腺病毒的构建和免疫原性

    Institute of Scientific and Technical Information of China (English)

    何芬; 靳晓宇; 邹俊涛; 牛道立; 杨俊华; 魏旋; 李建锋; 姚志彬

    2012-01-01

    目的 构建含有β-淀粉样蛋白( Aβ42)及核糖体进入位点(IRES)引导的粒细胞-巨噬细胞集落刺激因子(GM-CSF)双表达重组腺病毒疫苗.方法 PCR法扩增带有IgGK轻链信号肽序列的Aβ42基因、IRES基因和小鼠GM-CSF基因,定向克隆于穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒Ad-Aβ42-IRES-GMCSF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(AD-Aβ42).重组腺病毒经氯化铯密度梯度离心纯化后感染293细胞及Hela细胞,用免疫细胞化学和ELISA方法分别检测Aβ42及GM-CSF在细胞内和培养液中的分泌表达.免疫动物鉴定其免疫原性.结果 pAdTrack-Aβ42-GMCSF测序结果和预期一致,纯化的AD-Aβ42感染Hela细胞后,免疫细胞化学证实Aβ42基因在Hela细胞内表达.AD-Aβ42感染293细胞后,ELISA检测培养上清中Aβ42含量为(159.87±33.26)ng/mL,GM-CSF含量为(205.45±38.20)ng/mL.免疫动物能诱导机体产生高滴度的抗Aβ抗体.结论 成功制备了AD-Aβ42,核糖体进入位点(IRES)成功引导的GM-CSF的表达,感染细胞证实其呈分泌表达,接种动物能诱导高滴度抗Aβ抗体.为老年性痴呆(AD)的基因治疗做好铺垫.%Objective To construct coexpression adenovirus vaccine containing Aβ42 and GM-CSF guided by the internal ri-bosome entry site sequence (IRES) to prevent and therapy the Alzheimer' s Disease (AD). Methods The IgG K light chain fused Aβ42 sequence, IRES and the mouse GM-CSF gene were generated by PCR. And then they were cloned to the shuttle plasmid pAd-Track-CMV. The homologous recombination between the recombinant vector and the adenovirus bone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-Aβ42-IRES-GMCSF. The infecting recombinant virus particles AD-Ap42 was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293

  5. Safety and High Level Efficacy of the Combination Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP

    Science.gov (United States)

    Rampling, Tommy; Ewer, Katie J.; Bowyer, Georgina; Bliss, Carly M.; Edwards, Nick J.; Wright, Danny; Payne, Ruth O.; Venkatraman, Navin; de Barra, Eoghan; Snudden, Claudia M.; Poulton, Ian D.; de Graaf, Hans; Sukhtankar, Priya; Roberts, Rachel; Ivinson, Karen; Weltzin, Rich; Rajkumar, Bebi-Yassin; Wille-Reece, Ulrike; Lee, Cynthia K.; Ockenhouse, Christian F.; Sinden, Robert E.; Gerry, Stephen; Lawrie, Alison M.; Vekemans, Johan; Morelle, Danielle; Lievens, Marc; Ballou, Ripley W.; Cooke, Graham S.; Faust, Saul N.; Gilbert, Sarah; Hill, Adrian V. S.

    2016-01-01

    Background. The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector. Method. Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara–vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls. Results. No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected. Conclusions. The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types. Clinical Trials Registration. NCT01883609. PMID:27307573

  6. Boundaries of the origin of replication: creation of a pET-28a-derived vector with p15A copy control allowing compatible coexistence with pET vectors.

    Directory of Open Access Journals (Sweden)

    Sarmitha Sathiamoorthy

    Full Text Available During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1 to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+, and 2 to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+-derived plasmid with the p15A origin of replication (ori; pET-28a(+ contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+ replicon-comprising the ori, RNAI, RNAII, and Rom-with the p15A replicon, we generated pSAM, which contains the pET-28a(+ multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of

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

    Directory of Open Access Journals (Sweden)

    Jason S Richardson

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

  8. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease.

    Science.gov (United States)

    Sun, Chengqun; Gardner, Christina L; Watson, Alan M; Ryman, Kate D; Klimstra, William B

    2014-02-01

    Engineered alphavirus vectors expressing reporters of infection have been used for a number of years due to their relatively low costs for analysis of virus replication and the capacity to utilize imaging systems for longitudinal measurements of growth within single animals. In general, these vectors have been derived from Old World alphaviruses using a second viral subgenomic promoter to express the transgenes, placed either immediately after the nonstructural proteins or at the 3' end of the viral coding sequences. However, the relevance of these vectors to natural infections is questionable, as they have not been rigorously tested for virulence in vivo in comparison with parental viruses or for the retention of the reporter during replication. Here, we report construction of new expression vectors for two Old World arthritogenic alphaviruses (Sindbis and Chikungunya viruses) and two New World encephalitic alphaviruses (eastern and Venezuelan equine encephalitis viruses) based upon either fusion of the reporter protein in frame within nonstructural protein 3 (nsP3) or insertion of the reporter as a cleavable element between the capsid and PE2 structural proteins. We have compared these with a traditional 3' double subgenomic promoter virus expressing either a large, firefly luciferase (fLuc; 1,650 nucleotides), or small, NanoLuc (nLuc; 513 nucleotides), luminescent reporter protein. Results indicate that the nLuc is substantially more stable than fLuc during repeated rounds of infection regardless of the transgene location. However, the capsid-PE2 insertion and nsP3 fusion viruses exhibit the most authentic mimicking of parental virus infection regardless of expressed protein. IMPORTANCE As more antiviral therapeutics and vaccines are developed, rapid and accurate in vivo modeling of their efficacy will be required. However, current alphavirus vectors expressing reporters of infection have not been extensively tested for accurate mimicking of the infection

  9. Comparison of lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells%慢病毒载体和腺病毒载体转染原代培养的螺旋神经节细胞的比较

    Institute of Scientific and Technical Information of China (English)

    王国鹏; 罗凌惠; 谢静; 陈沛; 付勇; 钟刚; 龚树生

    2011-01-01

    Objective:We aimed to compare the characteristics between lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells (SG Cs). Method:SGCs from newborn rats were cultured and exposed to lentivirus-GFP and adenovirus-GFP vectors. GFP expression and the cell morphology were evaluated under epi-fluorescence microscope at 3 days and 7 days after exposure. Survival number of SGCs was counted, and the average percentage of SGCs with GFP expression was calculated, and axon length was measured by ImageJ software. Result:Cultured SGCs were transfected by either adenovirus or lentivirus vector successfully. The adenovirus vector presented an instant and efficient transfection. However, the expression of GFP went down after 7 days. In lentivirus-GFP group, GFP expression was detected at 7 days after exposure, and the number of cells with GFP expression increased gradually in the following days. Statistical analysis revealed that there were no differences in survival number of SGCs and average axon length among lentivirus-GFP group, adenovirus-GFP group and control group. Conclusion: Cultured SGCs can be transfected by either lentivirus vector or adenovirus vector safely and efficiently. SGCs are more susceptible to adenovirus vector, but GFP persists for a longer period after the lentivirus-mediated gene transfer.%目的:比较慢病毒载体和腺病毒载体转染新生大鼠耳蜗螺旋神经节细胞(SGCs)的不同转染特性.方法:体外培养新生大鼠SGCs,适当滴度的慢病毒载体和腺病毒载体转染SGCs,于病毒转染后3 d(培养后6 d)和转染后7 d(培养后10 d),荧光显微镜下观察绿色荧光蛋白报告基因(GFP)表达情况和SGCs的细胞形态.行SGCs计数,计算转染效率,并通过ImageJ软件测量各组SGCs轴突长度.结果:慢病毒载体和腺病毒载体均可有效转染体外培养的原代SGCs;腺病毒载体转染效率高,GFP表达快,而1周后GFP表达开始下降;慢病毒载体转染7 d

  10. Construction of a novel oncolytic adenoviral vector and its biological characteristics.

    Science.gov (United States)

    Zhang, Mingzhi; Zhang, Xudong; Han, Zhiqiang; Chen, Xinfeng; Yang, Li; Sheng, Yuqiao; Wen, Jianguo

    2013-02-01

    In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the

  11. Investigation of gene therapy of adenovirus in immune suppression

    Institute of Scientific and Technical Information of China (English)

    Xi XIA; Beibei WANG; Li CAO; Gang CHEN; Peng WU; Yunping LU; Jianfeng ZHOU; Ding MA

    2008-01-01

    The aim of this paper is to investigate the safety of reconstructed adenovirus in immunosuppressive ther-apeutics and to explore the role of ciclosporin A in ant-agonizing the elimination of the vector. Several rats were given retroperitoneal injection of purified ADV-TK in order to obtain models. After 14 days' treatment of ciclos-porin A, samples of different periods were obtained, then stained with hematoxylin-eosin (HE) to detect inflam-mation reactions. Immunohistochemistry was used to examine the expression of adenovirus in organs. The results are as follows: (1) In HE stained sections of the organs, some transitory and reversible inflammation was detected. (2) In immunohistochemistry assay, recon-structed adenovirus decreased gradually as time went by in the control group, while it did not happen in the experi-mental group in which the adenovirus showed a relative increase compared with their counterparts (P<0.05). (3) The distributions of adenovirus in the liver, spleen and lung were higher than those in the other organs detected. Reconstructed adenovirus as a vector is definitely safe in immunosuppressive therapeutics, and ciclosporin A, to some extent, is able to consequently inhibit the immune response of the rats and prolong the existing period of adenovirus.

  12. Construction of Txt5 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes%Txt5腺病毒载体构建及其在心肌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    彭浩; 陈宇; 李佑锋; 朱旋; 周作琼; 吴秀山; 范雄伟; 李永青

    2015-01-01

    设计小鼠 Txt5基因的特异性引物,将小鼠 cDNA 作为模板,用 PCR 扩增出 mTxt5编码区,并加入 HA标签序列.将这一片段插入 pMD -18T 载体,再亚克隆至 pAdtrack - cmv 穿梭载体上.线性化后,转化 BJ5183感受态细胞,在 BJ5183中发生同源重组获得 pAd - Txt5质粒.pAd - Txt5线性化后转染293A 细胞,包装得到含 Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过 RT - PCR 和免疫印迹法检测 HA 标签蛋白的表达.结果表明成功构建小鼠 Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.%Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein (GFP) was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .

  13. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Directory of Open Access Journals (Sweden)

    Marko T Ahonen

    Full Text Available BACKGROUND: Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. METHODS/RESULTS: We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. CONCLUSION: In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

  14. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    Science.gov (United States)

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  15. Chromatin structure of adenovirus DNA throughout infection

    OpenAIRE

    Giberson, Andrea N.; Davidson, Adam R.; Parks, Robin J.

    2011-01-01

    For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within...

  16. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter.

    Science.gov (United States)

    Li, Hua-Jung; Everts, Maaike; Pereboeva, Larisa; Komarova, Svetlana; Idan, Anat; Curiel, David T; Herschman, Harvey R

    2007-06-01

    Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to "retarget" the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can "retarget" adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases.

  17. 雄黄纳米颗粒在基因水平上抑制腺病毒复制的实验研究%Study on realgar nanoparticles inhibition of adenovirus replication at the gene level

    Institute of Scientific and Technical Information of China (English)

    王明哲; 甫尔哈提·吾守尔; 王成祥; 许文波

    2013-01-01

    Objective Modeling HAdV-3 infect Hep-2 cells in vitro.The effect of realgar nanoparticles on the expression of HAdV-3 is detected by using fluorescent quantitative PCR.Method The experiment is divided into four groups:Hep-2 cells control group,HAdV-3 virus control group,realgar nanoparticle group and ribavirin group.In order to detect HAdV-3 viral load,add realgar nanoparticles and ribavirin in vitro and remain that vitro for 24 hours when HAdV-3 has infected Hep-2 cells,extract total DNA of Hep-2 cells infected by HAdV-3,and establish Real-time PCR reaction system of every experimental groups.Result The Hep-2 cells group has no amplification curve,the Ct value is greater than 35,which illustrate HAdV-3 pathogen detection is negative.However,realgar nanoparticles group,ribavirin group and the HAdV-3 group have amplification curve,the Ct values are 29.30 ± 0.08,33.05 ± 1.29,26.01 ± 0.25 respectively,which illustrate HAdV-3 pathogen detection is positive.The viral copy amount of the adenovirus group(66 699 932 ±23.85) is more than that of realgar nanoparticles group (912 435.44 ± 16.57),and much greater than that of ribavirin group(459 124.84 ± 12.82) (P < 0.05).Conclusion The model of Hep-2 cell infected by HAdV-3 is reliable.The method of quantitative PCR is sensitive and specific.Realgar nanoparticles have a certain inhibition role for adenovirus nucleic acid replication.%目的 建立人腺病毒3型(HAdV-3)感染Hep-2细胞模型,采用荧光定量PCR(Real-time PCR)技术,观察雄黄纳米微粒在基因水平上对HAdV-3 DNA表达的影响.方法 将实验分为4组,即Hep-2细胞对照组、HAdV-3病毒对照组、雄黄纳米微粒组和利巴韦林组.在HAdV-3感染Hep-2细胞后,分别加入雄黄纳米微粒和利巴韦林作用24 h,提取HAdV-3感染Hep-2细胞的总DNA,建立Real-time PCR反应体系,测定各实验组HAdV-3的病毒载量.结果 Hep-2细胞对照组无扩增曲线,Ct >35,腺病毒病原体检测阴性;雄黄纳米微粒

  18. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

    Directory of Open Access Journals (Sweden)

    Akseli Hemminki

    2010-09-01

    Full Text Available Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

  19. Presence of protein at the termini of intracellular adenovirus type 5 DNA

    NARCIS (Netherlands)

    Wielink, P.S. van; Naaktgeboren, N.; Sussenbach, J.S.

    1979-01-01

    Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracel

  20. Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene%携带红色荧光蛋白报告基因的重组腺病毒载体的构建及其体外试验研究

    Institute of Scientific and Technical Information of China (English)

    卢建溪; 阳莉; 钱师宇; 王强

    2011-01-01

    目的:采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具.方法:将pTurboRFP-N上的含RFP基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N.用I-Ceu I/PI-Sce I双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5'040 pkGFP- II,回收目的片段,连接,获得重组腺病毒载体AdH5'.040.CMV.RFP-N.将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒.通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况.扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度.将所构建的重组腺病毒载体AdH5'.040.CMV.RFP-N与对照腺病毒载体AdH5.CMV.EGFP 在体外感染人肺癌细胞系A549 和乳腺癌细胞系MDA-MB-231.通过荧光蛋白RFP和EGFP的表达,比较它们对以上肿瘤细胞的感染效率.结果:经酶切鉴定,证明已正确构建重组腺病毒载体AdH5'.040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1015vp/L,滴度达1.8×1013pfu/L.重组腺病毒载体AdH5'.040.CMV.RFP-N感染人肺癌细胞系A549 和乳腺癌细胞系MDA-MB-231的感染效率,高于对照腺病毒载体AdH5.CMV.EGFP (P<0.05).结论:成功构建了携带RFP报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,对肿瘤细胞的感染效率高.RFP对GFP是一种很好的代替和补充,为基因治疗与基因疫苗研究提供更多的荧光标签.%AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP - N was digested and ligated into pShuttle transfer vector to

  1. Effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mDEF201 on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.

    Directory of Open Access Journals (Sweden)

    Donald F Smee

    Full Text Available An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201 was evaluated for efficacy against lethal cowpox (Brighton strain and vaccinia (WR strain virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl and pulmonary (50-µl, to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI, upper respiratory infections (URI, and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term or 56d (long-term prior to virus challenge. Single nasal sinus treatments of 10(6 and 10(7 PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201. Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7 mDEF201 treatments, but a 10(6 dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7 PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5 to 10(7 PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

  2. Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer.

    Science.gov (United States)

    Pulsinelli, G A; Temin, H M

    1991-09-01

    Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing splicing were large deletions, ranging in size from about 500 to about 2,000 bp. Most of the mutant proviruses lacked the encapsidation sequence, as shown by our inability to rescue the mutant proviruses with wild-type reticuloendotheliosis virus strain A and confirmed by Southern blotting and direct DNA sequence analysis. We therefore concluded that most of the deletions arose during reverse transcription in the target cell, rather than during transcription in the host cell. The sequence data also indicated that the deletions occurred by at least three different mechanisms: (i) misalignment of the growing point; (ii) incorrect synthesis and termination in the primer-binding sequence during synthesis of the plus-strand strong-stop DNA; and (iii) incorrect synthesis and termination before the primer-binding sequence during synthesis of the plus-strand strong-stop DNA. The second mechanism also led to the incorporation of cellular sequences into the proviral genome, pointing to a potential novel mechanism by which retroviruses can acquire cellular genes.

  3. Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV.

    Science.gov (United States)

    Tebas, Pablo; Stein, David; Binder-Scholl, Gwendolyn; Mukherjee, Rithun; Brady, Troy; Rebello, Tessio; Humeau, Laurent; Kalos, Michael; Papasavvas, Emmanouil; Montaner, Luis J; Schullery, Daniel; Shaheen, Farida; Brennan, Andrea L; Zheng, Zhaohui; Cotte, Julio; Slepushkin, Vladimir; Veloso, Elizabeth; Mackley, Adonna; Hwang, Wei-Ting; Aberra, Faten; Zhan, Jenny; Boyer, Jean; Collman, Ronald G; Bushman, Frederic D; Levine, Bruce L; June, Carl H

    2013-02-28

    We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

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

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    Lucile Tran

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

  5. Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

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    Tihana Jovanic

    Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

  6. Development of a Canine Adenovirus Type 1 Vaccine Strain E3-deleted Based Expression Vector%犬腺病毒1型疫苗株E3缺失表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    黎皓; 唐七义; 张云; 王树蕙; 郭彩云

    2001-01-01

    Objective To evaluate canine adenovirus type 1 vaccine strain (Cannaught Laboratory Limited,CLL) as recombinant vaccine and gene transfer vector. Methods Recombinant virus CLLEGFP which contains enhanced green fluorescent protein(EGFP) reporter gene was constructed. CLLEGFP was used to infect various human derived cell lines (293, Hela, CO, SW, Hep-2 and CAM) by inoculating intraperitoneally(IP), intravenously(IV)and intramuscularly (IM)to Kunming mice other than oral administration. Various tissue samples of the mice were collected at multitime point for observing EGFP green fluorescence. Anti-EGFP antibodies were detected by Western blot analysis in the sera after 4 weeks. Results CLLEGFP can infect various human derived cell lines and express EGFP. EGFP green fluorescence were observed in liver tissue cells after IP transducing 3 days. All immune inoculation ways above could induce Kunming mice producing anti-EGFP antibodies which were identified by Western blot analysis. Conclusions These resluts indicate that CLL possess powerful potential as recombinant vaccine and gene transfer vector.%探索以犬腺病毒1型疫苗株(Cannaught Laboratory Limited.CLL)作为病毒重组疫苗和基因转移载体的可行性。方法构建带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的E3缺失重组病毒CLLEGFP。将CLLEGFP感染各种人源细胞,并以灌胃、腹腔注射、尾静脉注射和肌肉注射等不同途径接种昆明小鼠。多时间点取小鼠组织标本,冷冻干燥切片,观察EGFP的表达。4周后采集小鼠血清,以Western blot分析抗EGFP 抗体的产生。结果 CLLEGFP能够感染各种人源细胞并表达EGFP。在腹腔接种CLLEGFP 3 d的小鼠肝组织细胞中可见转导的EGFP。Western blot分析显示,以各种途径免疫接种重组病毒4周后的小鼠血清中均存在抗EGFP特异抗体。结论 CLL具有开发成为病毒重组疫苗和基因转移载体的潜力。

  7. E1A genes of adenovirus type 2 and type 5 are expressed at different levels

    DEFF Research Database (Denmark)

    Moritz, Constanze; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses are an extensively studied system for modeling oncogenesis and for experimental cancer therapy. The most commonly analyzed virus types are 2 and 5, and little distinction has been made between them in past studies. Adenoviruses used for therapeutic purposes are frequently hybrids...... region. We found that the hybrid viruses replicated with considerably lower efficiency than their type 5 counterparts in H1299 cells (dl309:WtD = 3-4, dl338:dl1520 > 10). Moreover, adenovirus type 2 E1A expression from the hybrid viruses was strongly reduced in comparison to adenovirus type 5 E1A...

  8. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    Science.gov (United States)

    Khurana, Surender; Coyle, Elizabeth M; Manischewitz, Jody; King, Lisa R; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

  9. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    Directory of Open Access Journals (Sweden)

    Surender Khurana

    Full Text Available A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR. Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1 and heterologous A/Indonesia-5/2005 (clade 2.1 HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2 and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

  10. Crystallization and preliminary X-ray diffraction analysis of human adenovirus

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, V.S.; Natchiar, S.K.; Gritton, L.; Mullen, T.-M.; Stewart, P.L.; Nemerow, G.R. (Scripps); (Vanderbilt)

    2010-07-22

    Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in {approx}25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100 mM HEPES pH 7.0, 200 mM Ca(OAc){sub 2}, 14% PEG 550 MME, 15% glycerol in 100 mM Tris-HCl 8.5. Freshly grown crystals diffracted well to 4.5 {angstrom} resolution and weakly to 3.5 {angstrom} at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a = 854.03 {angstrom}, b = 855.17 {angstrom}, c = 865.24 {angstrom}, {alpha} = 119.57{sup o}, {beta} = 91.71{sup o}, {gamma} = 118.08{sup o} with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

  11. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    Science.gov (United States)

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  12. Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis

    Institute of Scientific and Technical Information of China (English)

    Ana Zeng; Tao Wang; Haiyang Xia; Shiyuan Peng; Weihua Chen; Chenglin Jiang; Lihua Xu

    2011-01-01

    The genus of Nocardiopsis is a new source of antibiotics,chemicals,and enzymes.Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation.By screening about 80 Nocardiopsis strains,6 of them harbored 8 plasmids (18-80 kb).The complete nucleotide sequence of pSQ10 consisted of 18,219 bp,with 71.9% G + Ccontent,encoding 17 open reading frames,5 of them resembled those of Streptomyces plasmids.A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp.YIM 90083,and rep protein bound to its iteron sequence.This system may be useful for gene cloning and expression in Nocardiopsis.

  13. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

    2007-01-01

    Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that e......Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin......-cell-independent immunity from adenovirus vectors offers prospects for vaccination against opportunistic pathogens in AIDS patients and possibly immunotherapy in chronic virus infections....

  14. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-02-01

    Full Text Available Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5 vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb.Methods: APC, a cationic polyethylene glycol derivative, was synthesized via two steps of ring-opening copolymerization of ethylene oxide and allyl glycidyl ether, followed by the addition of 2-mercaptoethylamine. The copolymer or the control PEI-2k was ionically complexed to anionic Ad5 in 5% glucose, and in vitro transduction assays were carried out in coxsackievirus and adenovirus receptor-positive cells (A549 and coxsackievirus and adenovirus receptor-negative cells (B16 and SKOV3. The physical properties and morphology of adenovirus alone or the complexes were investigated respectively by zeta potential, size distribution, and transmission electron microscopy image. Then cytotoxicity of APC was examined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Finally, the ability of APC to protect adenovirus from NAb was evaluated by transfection assays after a neutralizing effect.Results: APC was successfully synthesized and showed a low cytotoxicity. Positively charged Ad5/APC exhibited slightly increased diameter (130.2 ± 0.60 nm than naked Ad5 (115.6 ± 5.46 nm while Ad5/PEI-2k showed severe aggregation (1382 ± 79.9 nm. Ad5/APC achieved a gene transfection level as high as Ad5/PEI-2k in A549 or B16 cells, and significantly higher than Ad5/PEI-2k in SKOV3 cells. Most importantly, after the exposure to the neutralizing

  15. Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines.

    Directory of Open Access Journals (Sweden)

    Eric A Weaver

    Full Text Available All studies using human serotype 5 Adenovirus (Ad vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC, first-generation (FG and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines.

  16. A novel replication-competent vaccinia vector MVTT is superior to MVA for inducing high levels of neutralizing antibody via mucosal vaccination.

    Science.gov (United States)

    Huang, Xiaoxing; Lu, Bin; Yu, Wenbo; Fang, Qing; Liu, Li; Zhuang, Ke; Shen, Tingting; Wang, Haibo; Tian, Po; Zhang, Linqi; Chen, Zhiwei

    2009-01-01

    Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA) and a novel replication-competent modified vaccinia Tian Tan (MVTT) for inducing neutralizing antibodies (Nabs) via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S) of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels ( approximately 2-3-fold) of anti- SARS-CoV neutralizing antibodies (Nabs) than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (approximately 10-fold) higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination.

  17. A novel replication-competent vaccinia vector MVTT is superior to MVA for inducing high levels of neutralizing antibody via mucosal vaccination.

    Directory of Open Access Journals (Sweden)

    Xiaoxing Huang

    Full Text Available Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA and a novel replication-competent modified vaccinia Tian Tan (MVTT for inducing neutralizing antibodies (Nabs via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels ( approximately 2-3-fold of anti- SARS-CoV neutralizing antibodies (Nabs than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (approximately 10-fold higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination.

  18. HBsAg及B7-2抗原重组腺病毒载体感染免疫研究%Humoral immunization and cell-mediated immunization evoked by HBsAg and B7-2 Ag coexpression recombinant adenovirus vector

    Institute of Scientific and Technical Information of China (English)

    周智; 张定凤; 任红

    2001-01-01

    目的为激发机体对HBsAg的CTL反应,寻求对慢性乙型肝炎更有效的治疗方法。方法构建了共表达HBsAg及B7-2抗原的E1区插入重组腺病毒载体,用脂质体法转染293细胞,经空斑筛选表达目的抗原的重组腺病毒,用ELISA法及Western blotting法分别检测HBsAg及B7-2抗原表达。在293细胞中扩增,再纯化、浓缩后,体内感染C57小鼠,检测体液免疫反应和细胞免疫反应。结果重组腺病毒在体外培养的293细胞及HepG2细胞中高效表达HBsAg及B7-2抗原。感染小鼠体液免疫较弱,但可通过注射乙型肝炎疫苗而加强;重组腺病毒载体能诱导强而有效的细胞免疫反应;未观察到明显毒副作用。结论 HBsAg及B7-2抗原重组腺病毒载体体外感染293、HepG2细胞后高效表达目的抗原,感染免疫后诱导有效的细胞免疫反应。初步结果显示腺病毒载体是一种高效、安全的载体系统。%Objective To evoke cytotoxic T lymphocytes (CTL) response and seek for a more effective method to treat chronic hepatitis B. Methods The adenovirus vector was constructed with the foreign genes inserted in the early region 1(E1), which directed coexpression of HBV-S and B7-2 antigens by means of an internal ribosomal entry site placed between the two coding sequences. The vector was transfected into 293 cell lines by liposome and the adenovirus expressing the target antigens was obtained by plaque select. The HBsAg and B7-2 antigen expression in in vitro cell culture was measured by ELISA and Western blotting, respectively. The immune responses were measured by ELISA for antibody response and a LDH release assay for CTL activity after immunization with the recombinant adenovirus vector in C57 mice. Results HBsAg and B7-2 antigens were highly expressed after infecting the 293 and HepG2 cell lines in vitro. The humoral response to hepatitis B surface antigen was mildly induced and could be enhanced by reinjecting a

  19. Pharmacological Interventions for Improving Adenovirus Usage in Gene Therapy

    NARCIS (Netherlands)

    Haisma, Hidde J.; Bellu, Anna Rita

    2011-01-01

    Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction effici

  20. Pharmacological Interventions for Improving Adenovirus Usage in Gene Therapy

    NARCIS (Netherlands)

    Haisma, Hidde J.; Bellu, Anna Rita

    2011-01-01

    Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction

  1. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

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

    Directory of Open Access Journals (Sweden)

    Junji Uchino

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

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

    Science.gov (United States)

    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.

  4. The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

    Science.gov (United States)

    Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

    2012-12-15

    The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

  5. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  6. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yuanhui [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); He, Jinsheng, E-mail: jshhe@bjtu.edu.cn [College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Zheng, Xianxian [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wu, Qiang [Department of Pathology, Anhui Medical University, Hefei, Anhui, 230032 (China); Zhang, Mei; Wang, Xiaobo [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wang, Yan [Department of Pathology, Anhui Medical University, Hefei, Anhui, 230032 (China); Xie, Can; Tang, Qian; Wei, Wei [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wang, Min; Song, Jingdong; Qu, Jianguo [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); Zhang, Ying; Wang, Xin [College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); Hong, Tao [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China)

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  7. Construction of human 14-3-3 γadenovirus vector and its expression in PC12 cells%人14-3-3γ基因的腺病毒载体的构建及其在PC12细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    陈小武; 陈志斌; 王埮; 袁昆雄; 王淑荣; 孙圣刚

    2011-01-01

    Objective: To construct the recombinant adenovirus vector carrying human 14-3-3 γ gene, and infect the PC12 cells.Methods: The full 14-3-3 γ DNA sequence was obtained from plasmids Top1O/pHis-14-3-3 γusing PCR.The 14-3-3 γ gene was cloned into pAdTrack-CMV vector which was subsequently homologously recombined with pAdEasy-1 vector in the HEK293 cells to package the recombinant adenovirus vector (pAd-14-3-3 γ) carrying human 14-3-3 γ.After verified by PCR, we amplified pAd/14-3-3 γin HEK293 cells and purified it by CsCI gradient purification,titrated it using 50% tissue culture infective dose (TCID50) assay.Results: PC12 cells were infected with adenoviruses.Protein expressions of EGFP (enhanced green fluorescent protein) and 14-3-3 γwere detected by the intensity of green fluorescence under fluorescence microscope and Western Blot respectively.The 14-3-3 γgene was cloned and verified by sequencing and high tittered virus was produced by a construct carrying 14-3-3 γgene, and 14-3-3 γ was expressed efficiently in the PCI2 cells after infection.Conclusion: The newly constructed adenovirus vector containing human 14-3-3 γ gene provides the basis for genetherapy of Parkinson's disease.%目的:构建携带人14-3-3 γ基因的重组腺病毒表达载体并确定其对PC12细胞的感染效率.方法:采用PCR方法,从Top10/pHis-14-3-3 γ质粒中扩增14-3-3 γ DNA序列,将14-3-3 γ基因定向克隆到穿梭质粒载体pAdTrack-CMV,经与腺病毒骨架质粒pAdEasy-1载体同源重组后得到携带人14-3-3γ基因的重组腺病毒(pAd/14-3-3 γ),采用PCR的方法对重组腺病毒进行鉴定,转染HEK293细胞进行包装和扩增,氯化铯密度梯度离心法纯化,半数组织培养感染剂量(50%tissue culture infective dose,TCID50)方法测定重组腺病毒的滴度.体外感染PC12细胞,荧光显微镜观察绿色荧光蛋白(GFP)和Western Blot检测14-3-3γ蛋白的表达.结果:克隆得到人14-3-3γ基因,经PCR鉴定和测序证实

  8. Structure of Human Adenovirus

    OpenAIRE

    Nemerow, Glen R.; Phoebe L Stewart; Reddy, Vijay S.

    2012-01-01

    A detailed structural analysis of the entire human adenovirus capsid has been stymied by the complexity and size of this 150 MDa macromolecular complex. Over the past 10 years, the steady improvements in viral genome manipulation concomitant with advances in crystallographic techniques and data processing software has allowed structure determination of this virus by X-ray diffraction at 3.5 Å resolution. The virus structure revealed the location, folds, and interactions of major and minor (ce...

  9. Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats%Raf-1基因重组腺病毒 siRNA 载体构建及其对大鼠心肌细胞肥大的影响

    Institute of Scientific and Technical Information of China (English)

    郑亚萍; 刘春杰

    2016-01-01

    Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.%目的:构建特异性抑制大鼠Raf-1基因的重组腺病毒载体,并将其体外转导大鼠心肌细胞中进行功能鉴定。方法合成针对大鼠Raf-1的靶序列及阴性对照序列,经退火形成的DNA双链定向克隆到穿梭质粒pAdTrackCMV中获得pAdTrack-siRaf-1质粒,PmeI线性化后在BJ5183细菌中与pAdEasy-1骨架质粒进行同源重组获得pAd-siRaf-1质粒,后转染HEK293细胞,包装获得pAd-siRaf-1腺病毒颗粒,继而感染原代培养的心肌细胞,通过Western blot方法检测siRNA对Raf-1、NF-κB基因的抑制效率,液体闪烁计数仪测定3 H-亮氨酸([3 H

  10. Effect of recombinant adenovirus vector expressing human endostatin on endothelial cell proliferation%内皮抑素重组腺病毒表达载体构建及对内皮细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    唐辉; 徐永清; 李春晓; 张秀琼; 郑天娥; 刘旭盛; 梁晚益

    2008-01-01

    certain degree. OBJECTIVE: To construct a recombinant adenovirus vector expressing human endostatin (Ad/hEnd), and to investigate the cooperative effect of Ad/hEnd and keratinocyte on endothelial cell proliferation. DESIGN, TIME AND SETTING: Observational study, which was performed in the State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital of the Third Military Medical University of Chinese PLA between September 2006 and May 2007. MATERIALS: pAdTrack-CMV and pAdEasy-1 were obtained from Stratagene Company, USA; 293 cell and Ecoli.DH5α were stored in our laboratory. METHODS: The endostatin gene sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) based on mRNA of human fetal hepatic tissue and inserted into the adenovims shuttle plasmid pAdTrack-CMV to obtain recombinant plasmid pAdTrack-ES. After identification, positive recon was transformed into pAdeasy 1 recipient virus to screen positive clones. The adenovirus Ad/hEnd was generated from 293 cells and identified by PCR and fluorescence microscope. Then the keratinocytes were infected with Ad/hEnd, and co-cultured with endothelial cells by nest dish culture method. The content of endostatin was detected, and the non-transfection keratinocytes were used as the controls. MAIN OUTCOME MEASURES: Homologous recombination and identification of pAd/hEnd; generation and identification of Ad/hEnd; endostatin expression after 293 cell transfection; purification and titer measurement of Ad/hEnd; content of endostatin in culture solution; apoptotic percentage of endothelial cells; inhibitory ratio of endothelial cells. RESULTS: Ad/hEnd was constructed and the virus titer was generally up to 1.65×1012 PFU/L. Ad/hEnd-infected keratinocytes could effectively express and secrete endostatin of which the content reached 226 μg/L after 3 days of co-culture. The apoptotic percentage and inhibitory ratio of the

  11. 基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Carrying the Human Tissue Inhibitor of Metalloproteinase 1 cDNA

    Institute of Scientific and Technical Information of China (English)

    周毅; 屠冠军

    2012-01-01

    Objective To construct recombinant adenovirus vector carrying the human tissue inhibitor of metalloproteinase 1 (T1MP1) cD-NA and explore the feasibility of reversing or delaying intervertebral disc degeneration by gene therapy. Methods The hTIMPl cDNA was amplified from plasmid containing human TIMP1 ORF sequence by PCR. hTIMPl cDNA was subcloned into the adenovirus shuttle plasmid pDC316 of Ad5MaxTM adenovirus vector system,and the products were co-transfected into HEK293 cells with the helper plasmid pBH- Glox_El ,3Cre by lipofectin transfection method. The recombinant adenovirus Ad5-TIMP1 was generated by homologous recombination of the two plasmids in HEK 293 cells. After identification by PCR and enzyme digestion, Ad5-TTMP1 was amplified in HEK 293 adenoviral packaging cells, purified by column chromatography, and virus activity was measured by TCID50 assay. Results Recombinant adenoviral vector carrying hTIMPl(Ad5-hTIMPl) was successfully constructed with virus activity of l×l010 IU/ml,which was confirmed by PCR,restriction enzyme digestion and DNA sequencing. Conclusion The Recombinant adenoviral vector carrying hTIMPl (Ad5-hTlMPl) was successfully constructed, which provided a foundation for further research on reversing or delaying intervertebral disc degeneration by gene therapy.%目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析

  12. Crystal structure of human adenovirus at 3.5 Å resolution*

    OpenAIRE

    Reddy, Vijay S.; Natchiar, S. Kundhavai; Phoebe L Stewart; Nemerow, Glen R.

    2010-01-01

    Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here we report the X-ray structure at 3.5 Å resolution of the 150 megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber ...

  13. Effect of neutralizing sera on factor X-mediated adenovirus serotype 5 gene transfer

    NARCIS (Netherlands)

    Parker, A.L.; Waddington, S.N.; Buckley, S.M.K.; Custers, J.; Havenga, M.J.E.; Rooijen, N. van; Goudsmit, J.; McVey, J.H.; Nicklin, S.A.; Baker, A.H.

    2009-01-01

    The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated

  14. 用RNA干扰腺病毒载体研究Wnt/β-连环蛋白信号途径对人甲状腺细胞增殖的影响%Construction of GSK-3β-targeting RNAi adenovirus vector and the influence of Wnt/β-catenin pathway in proliferation of human thyrocytes

    Institute of Scientific and Technical Information of China (English)

    陈刚; 邹欣; 林丽香; 牟伦盼; 姚瑾; 游婷婷; 沈小燕; 朱香清; 乔玉芳; 林苗; 方晓文

    2008-01-01

    Objective To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3β (GSK-3β) and to observe its gene knockdown effect on the expression of GSK-3β, and to explore the effect of Wnt/β-catenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector. Methods An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3β gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney (HEK) 293A cells to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock . Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3β specific RNAi adenovirus. The GSK-3β gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups: infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of β-catenin. Results The GSK-3β expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST (all P <0.05). The expression of β-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group (both P<0.05). BrdU assay suggested that the proliferation rates 1,3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST (all P<0.05). Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently

  15. 人Nanog基因重组腺病毒载体的构建及其在人脐带间充质干细胞中的表达%Construction of recombinant adenovirus vector carrying human Nanog gene and its expression in human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    孙靖; 徐哲; 钱茜; 严家来; 陈圆; 张徐; 高硕; 钱晖; 许文荣

    2011-01-01

    Objective To construct recombinant adenovirus vector carrying human Nanog gene and transfect human umbilical cord mesenchymal stem cells (hucMSCs). Methods The amplification products of Nanog in polymernse chain reaction (PCR) by using a pair of primers containing the sites of restriction endonuclease Kpn I and Xho I were subcloned into shuttle plasmid pAdtrack-CMV.After analysis of restriction endonuclease and confirmation by sequencing, the recombinant shuttle plasmid pAdTrack-CMV-Nanog was linearized by Pme I , and then transformed into E. coli. BJ5183 which was transformed by adenoviral backbone plasmid pAdEasy-1.The recombinant plasmid pAd-Nanog obtained from screening was confirmed by PCR and restriction endonuclease analysis. The pAdNanog plasmid was linearized by Pac Ⅰ and transfected into human embryonal kidney cell 293A via liposome. The recombinant adenovirus was packaged and amplified in 293A cells following three amplification. The prepared highly expresed Ad-Nanog was transfected into hucMSCs. Results PCR and restriction endonuclease analysis confirmed that Nanog gene was inserted into the recombinant adenovirus vector successfully. The efficiently expressed green fluorescent protein (GFP) in transfected hucMSCs was visualized by fluorescent microscopy. Conclusion Recombinant adenovirus vector containing Nanog was constructed successfully and efficiently transfected hucMSCs which should be useful in the successive investigation on transgenic mesenchymal stem cells.%目的 构建重组人Nanog基因腺病毒栽体(Ad-Nanog),转染人脐带间充质干细胞(hucMSCs),用于后续研究.方法 设计含有Kpn Ⅰ及Xho Ⅰ酶切位点的引物,PCR扩增Nanog基因,将扩增产物亚克隆到pAdTrack-CMV穿梭质粒上,经双酶切和基因测序鉴定,重组穿梭质粒经Pine Ⅰ线性化后,在含有腺病毒骨架质粒pAdEasy-1的BJ5183中同源重组,筛选获得Ad-Nanog重组腺病毒质粒.经Pac Ⅰ酶切线性化,脂质体转染293A细胞,包

  16. 大鼠SOCS3基因重组腺病毒载体的构建及转染血管平滑肌细胞后的表达%Construction of Rat SOCS3 Recombinant Adenovirus Vector and Its Expression in Rat Primary Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    向水; 董念国; 刘金平; 史嘉玮; 肖雅琼; 王玉

    2012-01-01

    目的 构建携带大鼠SOCS3基因的重组腺病毒载体并转染大鼠血管平滑肌细胞,评价其转染效果,为血管增殖性疾病转基因治疗的实验研究奠定基础.方法 用BamH Ⅰ和EcoR Ⅰ将目的基因pUC57-Simple-rSOCS3及腺病毒穿梭载体pYr-adshuttle- 4(带GFP标记)行双酶切.回收酶切质粒的目的片段进行连接,产物转化至DH5α感受态.提取质粒酶切鉴定正确后测序.通过LR体外同源重组将rSOCS3表达框构建至pAd/PL-DEST腺病毒表达载体上.将腺病毒线性化后转染HEK293细胞包装腺病毒,PCR鉴定此腺病毒中是否含有rSOCS3基因.放大培养并收集病毒,TCD法测定病毒滴度.将此病毒感染大鼠血管平滑肌细胞,荧光显微镜观察感染效率,real-time PCR及免疫印迹检测SOCS3 mRNA及蛋白表达.结果 rSOCS3腺病毒穿梭质粒酶切鉴定正确,测序与rSOCS3基因库中的序列完全相符.PCR鉴定pAd/PL-DEST腺病毒表达载体成功携带目的基因rSOCS3.TCD法测定的最终滴度为2×1010pfu/mL.荧光显微镜观察此病毒感染血管平滑肌细胞的效率在80%以上,real-time PCR及免疫印迹检测结果显示血管平滑肌细胞中SOCS3 mRNA及蛋白表达明显上调.结论 成功构建携带大鼠SOCS3基因的腺病毒载体,并在血管平滑肌细胞中高表达,为血管增殖性疾病转基因治疗的实验研究奠定了基础,具有一定的应用价值.%Objective To construct the recombinant adenovirus vector carrying rat SOCS3 gene and assess its transfection efficiency in vascular smooth muscle cells(VSMCs). Methods The rat SOCS3 plasmid(pUC57-Simple-rSOCS3)and advenovir-us shuttle plasmid(pYr-adshuttle-4)containing green fluorescent protein(GFP)reporter gene were cleaved by restriction endonu-clease BamW I and EcoR I . The target gene fragments were connected together to generate a recombinant plasmid pYr-ad-shuttle-4-rSOCS3 that was transfected into E. coli DH5a. The plasmid was confirmed to be constructed as

  17. CEA启动子启动双自杀基因CD/TK重组AdEasy XL腺病毒构建%The construct of replication deficient AdEasy XL adenovirus containing CEA promoter, CD and TK gene

    Institute of Scientific and Technical Information of China (English)

    王天宝; 汪建平; 董文广; 钟女奇; 周军

    2005-01-01

    目的构建含有CEA启动子(CEA promoter,Cp)启动的双自杀基因CD与TK的复制缺陷型腺病毒.方法设计带有限制性内切酶酶切序列的PCR引物.在2μl T4 DNA ligase作用下,Cp插入pAdTrack(pAT)的MCS,形成pAT.Cp;CD插入pAT.Cp的MCS,形成pAT.Cp.C;TK插入pAT.Cp.C的MCS,形成pAT.Cp.C.T.5 μg pAT.Cp.C.T经4 μl Pme Ⅰ线化后,转化BJ5183-AD-1,1μl PacⅠ酶切鉴定.重组AdEasy质粒转化XL10-Gold细胞,大量扩增提取纯化,转染5 × 105 AD-293细胞,荧光显微镜观察,制备带有Cp.C.T重组腺病毒(recombinant adenovirus with Cp.C.T,RA-Cp.C.T),测定病毒滴度.结果pAT.Cp,pAT.Cp.C,pAT.Cp.C.T双酶切以及PCR均见长约500 bp的Cp,1 300 bp的CD,1 100 bp的TK.对照质粒DNA转化BJ5183-AD-1效率为7.36×106 cfu/μg.线化pAT.Cp.C.T转化BJ5183-AD-1,形成重组AdEasy质粒DNA,Pac Ⅰ酶切见典型长约3.0或4.5kb与30.0kbDNA片断.重组质粒转染AD-293出现绿色荧光.RA-Cp.C.T滴度为5.67×107 pfu/ml.结论RA-Cp.C.T构建正确;AdEasy XL腺病毒载体系统转导Cp.C.T具有高效性,易于观察转染效果.

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

    Directory of Open Access Journals (Sweden)

    Carmen Unzu

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

  19. Challenges in manufacturing adenoviral vectors for global vaccine product deployment.

    Science.gov (United States)

    Vellinga, Jort; Smith, J Patrick; Lipiec, Agnieszka; Majhen, Dragomira; Lemckert, Angelique; van Ooij, Mark; Ives, Paul; Yallop, Christopher; Custers, Jerome; Havenga, Menzo

    2014-04-01

    Abstract Once adenovirus vector-based vaccines are licensed for the prevention of important infectious diseases, manufacturing processes capable of reliably delivering large numbers of vaccine doses will be required. The highest burden of disease for many infectious pathogens under investigation occurs in resource-poor settings. Therefore, the price per dose will be an important determinant of success. This review describes common practices for manufacturing replication-incompetent adenovirus vectors at clinical scale. Recent innovations and strategies aimed at improving the cost-effectiveness of manufacturing and ensuring high-volume vaccine production and purification are described. Hereto, technologies to increase bioreactor yields are reviewed. In addition, the use of single-use perfusion bioreactors, modification of some purification steps to avoid the use of expensive endonucleases, and use of charged filters during anion exchange all have the potential to bring down the cost of goods and are thus described. Finally, processes for ensuring quality throughout the manufacturing process, methods for testing viral identity, and safety of master seeds through to the end vaccine product are described.

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

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of); Kim, Yeon Soo [Schoole of Medicine, Inje University, Seoul (Korea, Republic of)

    2004-07-01

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

  1. 构建含人胰岛素样生长因子基因腺病毒载体及在兔骨髓间充质干细胞的表达%Construction of adenovirus vectors containing human insulin-like growth factor-1 gene and its expression in rabbit mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    丁幸坡; 金先庆; 罗小辑; 邱林; 刘伟

    2008-01-01

    transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

  2. Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice.

    Science.gov (United States)

    Maunder, Helen E; Taylor, Geraldine; Leppard, Keith N; Easton, Andrew J

    2015-11-27

    Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8(+) T cell response and, to a lesser extent, a CD4(+) T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.

  3. Adenovirus-based p53 gene therapy in ovarian cancer.

    Science.gov (United States)

    Santoso, J T; Tang, D C; Lane, S B; Hung, J; Reed, D J; Muller, C Y; Carbone, D P; Lucci, J A; Miller, D S; Mathis, J M

    1995-11-01

    Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.

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

    Directory of Open Access Journals (Sweden)

    Shakti Singh

    Full Text Available 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.

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

    Science.gov (United States)

    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

  6. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲

    2016-01-01

    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  7. Adenovirus infection in immunocompromised patients

    Directory of Open Access Journals (Sweden)

    Sylwia Rynans

    2013-09-01

    Full Text Available Human adenoviruses belong to the Adenoviridae family and they are divided into seven species, including 56 types. Adenoviruses are common opportunistic pathogens that are rarely associated with clinical symptoms in immunocompetent patients. However, they are emerging pathogens causing morbidity and mortality in recipients of hematopoietic stem cell and solid organ transplants, HIV infected patients and patients with primary immune deficiencies. Clinical presentation ranges from asymptomatic viraemia to respiratory and gastrointestinal disease, haemorrhagic cystitis and severe disseminated illness. There is currently no formally approved therapy for the treatment of adenovirus infections.This article presents current knowledge about adenoviruses, their pathogenicity and information about available methods to diagnose and treat adenoviral infections.

  8. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    Science.gov (United States)

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia

    2016-10-01

    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

  9. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    Science.gov (United States)

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  10. Adenovirus-based vaccine against Listeria monocytogenes

    DEFF Research Database (Denmark)

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

    2013-01-01

    The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC...... class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular...... bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP...

  11. mIL-18腺病毒载体构建及在大鼠骨髓间充质干细胞中的表达%Construction of adenovirus vectors contained mIL-18 and expression in rat mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    许刚; 郭燕舞; 徐如祥; 姜晓丹

    2012-01-01

    目的 构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础.方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化.体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达.结果 成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达.结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad- mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞.%Objective To construct adenovirus vectors contained mIL-18 and observation the expression in rBMSCs and explore experiment study for gene therapy of gliorna. Methods mIL-18 gene was subcloned into the shuttle plasmid pAdtrackCMV, then linearized the vector and convert competent cell BJ5183 which contain the adenovirus frame plasmid pAdEasy-1. The homologous recombination plasmid was packaged by HEK293 cells and purified by CsCL The cell im-munophenotype was detected by flow cytometry. The expression of mIL-18 was measured by fluorescence microscope. RT-PCR and ELISA. Results The adenovirus vector Ad- mIL-18 was successfully constructed, which was labeled by green fluorescent protein (GFP) and effectively expressed in rBMSCs, Conclusion pAdEasyl is an effective gene trans-fection system. rBMSCs transfected with Ad- mIL-18 can serve as the seeds cells for the gene therapy of glioma.

  12. pri-miR-21重组腺病毒载体构建及其对TLR4基因调控的研究%Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4

    Institute of Scientific and Technical Information of China (English)

    赵婧; 徐广贤; 贾伟; 董辉; 张一琳; 赵志军; 魏军

    2012-01-01

    目的:构建能高效表达成熟miR-21小分子的腺病毒载体,探讨其对TLR4基因的靶向调控关系.方法:以正常小鼠基因组DNA为模板,PCR扩增pri-miR-21基因,克隆至穿梭载体pAdTrack-CMV中经PCR、酶切及基因测序分析正确无误后,与pAdEasy-1腺病毒骨架质粒进行同源重组,并利用293A细胞,包装成pri-miR-21重组腺病毒感染HeLa细胞,通过Westem blot检测TLR4的蛋白表达水平,验证miR-21与TLR4靶向调控的关系.结果:经PCR、酶切、测序及GFP表达证实,成功构建携带pri-miR-21基因的重组腺病毒载体并制备出高滴度重组病毒.Western blot证实,miR-21可抑制TLR4蛋白的表达.结论:所制备的小鼠pri- miR-21重组腺病毒,可以高效表达成熟miR-21小分子,能够抑制靶基因TLR4的表达.%AIM: To construct the recombined adenovi-rus 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. METHODS: 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. RESULTS; Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioin-formatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. CONCLUSION-. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  13. A targeting ligand enhances infectivity and cytotoxicity of an oncolytic adenovirus in human pancreatic cancer tissues.

    Science.gov (United States)

    Yamamoto, Yuki; Hiraoka, Nobuyoshi; Goto, Naoko; Rin, Yosei; Miura, Kazuki; Narumi, Kenta; Uchida, Hiroaki; Tagawa, Masatoshi; Aoki, Kazunori

    2014-10-28

    The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

  14. Construction and characterization of a hexon-chimeric human adenovirus type 3 vector expressing one major epitope of dengue virus type 1%1型登革病毒抗原表位嵌合人3型腺病毒六邻体重组病毒的构建及免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    招穗珊; 周志超; 李潇; 樊晔; 廖小红; 周荣; 苏晓波

    2015-01-01

    Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.%目的:构建六邻体嵌入1型登革病毒( DENV1)抗原表位的人3型重组腺病毒,鉴定其抗原性。方法以人3型腺病毒骨架质粒pBRAdΔE3GFP为模板,overlap PCR在六邻体高变区HVR1插入DENV1的抗原表位,突变的六邻体片段克隆到穿梭载体,酶切后与线性化的3型腺病毒骨架质粒pBRAdΔE3GFP在大肠杆菌BJ5183同源重组,获得阳性重组腺病毒质粒pBRAdΔE3GFP-DENV1。线性化后转染AD293细胞拯救重组腺病毒rAdΔE3GFP-DENV1并大量培养。纯化后腺病毒免疫BALB/c小鼠,通过ELISA和Western blot检测小鼠的体液免疫应答。结果在人3型腺病毒六邻体成功插入DENV1抗原表位并包装出重组腺病毒,ELISA和Western blot结果显示小鼠免

  15. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

  16. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

    Directory of Open Access Journals (Sweden)

    Anton V Borovjagin

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

  17. Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

    Science.gov (United States)

    Thorne, Barbara A; Quigley, Paulene; Nichols, Gina; Moore, Christine; Pastor, Eric; Price, David; Ament, Jon W; Takeya, Ryan K; Peluso, Richard W

    2008-01-01

    Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

  18. Future prospects for the development of cost-effective Adenovirus vaccines

    DEFF Research Database (Denmark)

    Fougeroux, Cyrielle; Holst, Peter J

    2017-01-01

    Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more efficient and cost-effective vaccines that would improve access to those in need. In this review, we will describe the status of virus......-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient...... as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes...

  19. 表达人JNK基因重组腺病毒的构建和鉴定%Construction and identification of expressing human c-Jun N-terminal kinase(JNK)recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    陈金虎; 刘慧霞; 张佳妮; 郭敏; 全养雅; 谭莺

    2008-01-01

    Objective To construct replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase by homologous recombination.Methods The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus,and then the recombinant adenovirus was detected by PCR and DNA sequencing.Results JNK recombinant adenoviral vector was effectively transfected into HEK 293 cells and was successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)was observed on the 5th day after transfection.The fragment of JNK gene was amplified by PCR and identified by sequencing.The animal experiment confirmed that Ad-WT-JNK was effectivety expressed in liver tissue. Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.And the achievement laid a foundation for further investigation of the function and application of JNK.%目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒.方法 将重组穿梭载体pAdTrack-CMV-WT-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.结果 JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定.动物实验证实构建的Ad-WT-JNK能有效在肝组织表达.结论 该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒,为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础.

  20. A novel and simple method for construction of recombinant adenoviruses.

    Science.gov (United States)

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin

    2006-07-19

    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

  1. Inhibitory effects of conditionally replicative adenovirus armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control on human renal carcinoma OSRC cells%荷载管家基因shRNA的双调控溶瘤腺病毒抑制肾癌细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    李连涛; 张宝福; 李慧忠; 郑骏年

    2012-01-01

    Objective To study the effects of suppressing cell proliferation and GAPDH gene expression on human renal carcinoma OSRC cells with TD - GAPDH, a conditionally replicative adenovirus ( CRAds ) armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control . Methods Renal carcinoma OSRC cells and human proximal tubular epithelial cells ( HK - 2 ) were infected with TD - GAPDH, TD55, ZD55 - EGFP. The protein expressions of E1A were detected by Western blot. GAPDH expressing levels of OSRC cells were detected by RT -PCR and Western blot. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTT method. OSRC cells were stained with crystal violet to detect cytotoxici effect . Results Western blot assay of E1A expression indicated OSRC cells infected with TD - GAPDH, TD55, ZD55 - EGFP expressed E1A, not detected E1A protein expression in HK - 2 cells. Significant reduction of GAPDH mRNA and protein content was observed in the lysates from OSRC cells in -fected with TD - GAPDH. TD - GAPDH treatment of OSRC cells resulted in increased apoptotie cell death than TD 55 and ZD55 - EGFP treatment. Results of crystal violet stain and MTT assay demonstrated that the antitumor effect of TD-GAPDH was more potent than both TD55 and ZD55 - EGFP. Conclusion CRAds armed with shRNA against GAPDH gene could duplicate in tumor cells , which has both the lytic ability of oncolytic adenovirus and the capacity to deliver shR -NA. TD - GAPDH armed with shRNA gene can significantly inhibit tumor cell growth , which may provide a new platform for cancer gene therapy with shRNA and mayprove to be a useful novel tool for renal cancer therapy .%目的 研究荷载管家基因GAPDH shRNA的溶瘤腺病毒(TD-GAPDH)对人肾癌细胞OSRC增殖抑制的作用.方法 病毒TD-GAPDH、TD55、ZD55-EGFP感染OSRC细胞、人肾小管上皮细胞HK-2.Western blot法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法分别检测GAPDH

  2. Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate.

    Science.gov (United States)

    Lee, C T; Seol, J Y; Park, K H; Yoo, C G; Kim, Y W; Ahn, C; Song, Y W; Han, S K; Han, J S; Kim, S; Lee, J S; Shim, Y S

    2001-01-01

    High frequency of p16 alteration and high local recurrence rate of bladder cancer make this cancer an ideal target for p16 gene therapy. However, a low transduction rate of p16 via adenoviral vector causes an inconsistent result. In this study, we have tested adenovirus-p16 in several bladder cancer cell lines and investigated a way of improving the low transduction rate. Adenovirus-p16 showed a strong antitumor effect on bladder cancer cell lines (253J and T24) with strong Coxackie-adenoviral receptor (CAR) expression but little antitumor effect on bladder cancer cell lines (J82 and HT1376) with little CAR expression. In this study, we suggest a simple way of overcoming the differential effects of the adenovirus. The addition of butyrate to media was found to increase the transduction rate of adenovirus remarkably and increase the antitumor effect of adenovirus-p16 in bladder cancer cell lines with little CAR expression. Butyrate effects were related with increased CAR expression on the cell surface as well as increased transgene expression from adenoviral vector. From these observations, application of adenovirus-p16 gene therapy with butyrate can overcome the obstacle of low gene transfer and enhance the antitumor effect of adenovirus-p16 in bladder cancer.

  3. 重组GGPPS腺病毒载体的构建及其调节肝星状细胞活化的研究%Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs

    Institute of Scientific and Technical Information of China (English)

    来珊珊; 陈卫波; 徐康; 丁尧; 沈宁; 潘飞燕; 李朝军; 薛斌

    2011-01-01

    目的:构建携带人源ggpps基因的重组腺病毒并用其研究GGPPS对人肝星状细胞的活化作用.方法:利用PCR方法扩增ggpps基因片段,并亚克隆至腺病毒穿梭质粒pAdTrack-CMV中,形成穿梭质粒pAdTrack-CMV-GGPPS.测序正确后,经PmeⅠ单酶切线性化的pAdTrack-CMV-GGPPS与腺病毒骨架质粒pAdEasy-1用电穿孔法共转化大肠杆菌BJ5183,进行同源重组.经PacⅠ酶切鉴定正确后,用磷酸钙法转染QBI-293A细胞,包装成重组体腺病毒Ad-GGPPS颗粒.利用穿梭质粒pAdTrack-CMV中GFP报告基因的表达,观察重组腺病毒的产生.用TCID50法测定病毒颗粒的浓度,并用免疫蛋白印迹和实时荧光定量PCR方法鉴定目的基因的表达.然后利用该病毒感染人肝星状细胞LX-2,观察对肝星状细胞的活化以及细胞外基质合成的影响.结果:成功地构建了携带ggpps基因的重组腺病毒,得到的病毒滴度经TCID50法检测为2.5×109 PFU/mL.该病毒能够在人源的肝脏细胞中成功表达GGPPS蛋白.在LX-2细胞中过量表达GGPPS可以促进肝星状细胞(Hepatic stellate cells,HSCs)转分化成表达平滑肌肌动蛋白α(Alpha-Smooth muscle actin,α-SMA)的肌成纤维细胞(Myofibroblast,MF),同时检测到胞外基质成分纤连蛋白( Fibronectin,FN)的表达量显著增加.结论:成功构建的重组GGPPS腺病毒能促进肝星状细胞的活化.%Objective: To construct the recombinant adenovirus vector containing human ggpps gene and study its effect on activation of HSCs. Methods: ggpps gene was obtained by PCR amplification and subcloned into adenovirus shuttle plas-mid of pAdTrack-CMV to form the shuttle vector of pAdTrack-CMV-GGPPS. After sequencing, it was linearized with Pmel and co-transfected into E. Coli BJ5183 cells with adenovirus genomtc plasmid of pAdEasy-1 to achieve homologous recombination. The DNA of identified recombinant plasmid was digested with Pacl and then transfected into QBI-293A cells to package adenovirus

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-15

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

  5. Construction and identification of replication recombinent adenovirus vector of mice LINGO-1 shRNA%含鼠LINGO-1 shRNA重组腺病毒载体的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    高峰; 孙红辉; 杨有庚

    2009-01-01

    目的 构建含鼠LINGO-1短发夹RNA(short hairpin RNA,shRNA)的复制缺陷型腺病毒载体.方法 通过PCR法将带有LINGO-1 shRNA序列构建至U6启动子(U6sh LINGO-1)下游,与T载体连接,将质粒转化入大肠杆菌.将U6sh LINGO-1连到穿梭质粒pDC316上,与腺病毒基因组质粒pBHGlox△E1、3Cre共转染293T细胞,得到重组腺病毒载体(rAd LINGO-1).经PCR鉴定正确后,进行扩增、纯化、滴度测定及感染性鉴定.结果 PCR法得到U6sh LINGO-1,rAd LINGO-1具有良好的感染性,腺病毒滴度可达109TCID50/ml.双引物PCR法鉴定Ad LINGO-1可扩增出Ad及特异性片段U6sh LINGO-1.结论 成功构建了能表达LINGO-1 shRNA的重组腺病毒载体rAd LINGO-1,可能为临床应用RNA干涉技术促进脊髓损伤的恢复提供新的方法.

  6. Adenovirus Type 11 Uses CD46 as a Cellular Receptor

    OpenAIRE

    Segerman, Anna; Atkinson, John P.; Marttila, Marko; Dennerquist, Veronica; Wadell, Göran; Arnberg, Niklas

    2003-01-01

    The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type...

  7. HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy

    Directory of Open Access Journals (Sweden)

    Xu Hai-Neng

    2011-11-01

    Full Text Available Abstract Background In previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947 and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24.ΔE1B (briefly Ad.SPDD. HCCS1 (hepatocellular carcinoma suppressor 1 is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer. Results Ad.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion. Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway. Conclusions These results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

  8. 去甲基化酶腺病毒载体构建及其在人颌下腺细胞中的表达%Cloning and expression of methyl-CpG binding domain 2 gene in human submandibular gland cell line by adenovirus vector transfection

    Institute of Scientific and Technical Information of China (English)

    李莹; 刘淑丹; 杨彦春; 董世武; 熊剑; 李方; 周继祥

    2011-01-01

    目的 构建去甲基化酶(methyl-CpG binding domain 2,MBD2)基因腺病毒载体,并观测其在人颌下腺(human submandibular gland,HSG)细胞中的表达.方法 以HSG细胞总RNA为模板,RT-PCR法扩增MBD2基因全长编码序列,克隆入载体pMD18-T后,亚克隆入pAdTrack-CMV腺病毒穿梭载体,构建pAdTrack-MBD2重组体.该重组体与腺病毒骨架质粒pAdEmy-1于BJ5183菌中同源重组,产生重组腺病毒质粒Ad-MBD2,该质粒经293细胞包装,获得具有感染力的Ad/MBD2重组腺病毒颗粒,将该病毒颗粒感染HSG细胞,倒置显微镜检测转染后HSG细胞生长变化及MBD2在其中的表达等情况.图像分析法计算感染效率.RT-PCR法检测MBD2基因在该细胞中的表达变化.结果 成功克隆到909 bp大小的MBD2基因,构建了其腺病毒载体Ad/MBD2,该腺病毒载体经293细胞成功包装,并获具感染力的Ad/MBD2重组腺病毒颗粒,其感染效率约70%.感染的HSG高表达MBD2.结论 成功克隆到MBD2基因,构建了其重组腺病毒载体Ad/MBD2,并证实了其感染HSG细胞的有效表达.%Objective To construct the adenovirus vector carrying methyl-CpG binding domain 2 (MBD2) gene and express this gene in human submandibular gland (HSG)cell line. Methods cDNA fragment encoding human MBD2 gene was amplified by reverse transcriptase-polynierase chain reaction (RT-PCR) with the total mRNA isolated from HSG cells as template. The PCR amplified product was first cloned into pMD18-T vector, and then subcloned into the shuttle vector pAdTrack-CMV in order to construct the recombi-nant plasmids pAdTrack-MBD2, which homologously recombinated with the adenoviral backbone vectors Adeasy-1 in BJ5183 bacterial cells, so as to generate recombinant adenoviral plasmids Ad-MBD2. The recombi-nant adenoviruses Ad/MBD2, which were employed to infect HSG cells, were generated by transfecting the recombinant adenoviral DNA into 293 cells. The growth of transfected HSG cells and the expression of enhanced

  9. Sensitization of prostate cancer cell lines to 5-fluorocytosine induced by a replication incompetent adenoviral vector carrying a cytosine deaminase transcription unit

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM: To investigate the efficiency of cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines. METHODS: Cell culture, infectivity test and sensitivity test, observing the bystander effect and animal model experiment were carried out. RESULTS: All the established prostate cancer cell lines were eventually infectable, but ratio of vector/cell and time of exposed at which infection occurs was dependent on the cell lines. The expression of transfered cytosine deaminase gene peaked at different days, but persisted beyond 11 days. The prostate cell lines were sensitized to the 5-fluorocytosine by infection with the cytosine deaminase gene adenoviral vector, and only 5% of the LNCap and 10% of the RM-1 cells infected were required for 100% cell death. In the animal model, there was significant eradiation of tumor growth at the ratio of 400 vector particles/cell and with the systematic treatment of 5-fluorocytosine. CONCLUSION: The adenoviral vector carrying a cytosine deaminase transcription unit can sensitize the prostate cancer cell lines to 5-fluorocytosine, and the system can significantly inhibit the growth of prostatic tumor in mice.

  10. Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    CHEN Ji-quan; GAO Xue-tao; XIU Qing-yu; YU Yi-zhi; LUO Wen-tong

    2001-01-01

    To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-18 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1)IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus, and the concentration of IL-18 in lung lavage was higher than that in pelipheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on experimental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL-18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-05-01

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

  12. 牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达%Construction of bovine Neospora caninum NcSRS2 adenovirus shuttle vector and expression of NcSRS2 gene in 293 cells

    Institute of Scientific and Technical Information of China (English)

    贾立军; 张守发; 刘明明

    2012-01-01

    应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2(AF061249)核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。%In this research,bovine Neospora caninum NcSRS2 gene was amplified by PCR.The purified PCR products were ligated with pMD18-T simple vector.After enzyme digestion and sequence analysis,the correct pMD-18T-NcSRS2 gene was digested by EcoRⅠand XbaⅠ,and cloned into the adenovirus shuttle vector pCR259 which was digested by the same enzymes.The correct pCR259-NcSRS2 recombinant plasmid was transfected into 293 cells.The expression of recombinant plasmid in 293 cells was detected by IFAT and Western-blotting.The result shows that the length of bovine Neospora caninum NcSRS2 gene is 1 227 bp.which has 99% homology with sequence and sequence in GenBank.The pCR259-NcSRS2 recombinant plasmid could transiently express in 293 cells.The molecular weight of the expressed protein which has good reactionogenicity is 43 000.This research laid the foundation for the construction of Neospora caninum vector-adenovirus vaccine

  13. Co-application of ricin A chain and a recombinant adenovirus expressing ricin B chain as a novel approach for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Hong-bin WANG; Fei XIA; Jing GE; Juan YIN; Li-song TAN; Pei-de ZHANG; Jiang ZHONG

    2007-01-01

    Aim: To develop a novel ricin-based approach for the safe and effective therapy of cancer. Methods: The ricin A chain (RTA) was expressed in Escherichia coli in the form of a 6xHis-tagged fusion protein and purified with Ni2*-NTA affinity resin. A replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702. Cell viability was determined with trypan blue staining or MTT assay. Results: The expression and release of RTB, as well as the entry of RTA into AdGFP-RTB-infected cells were confirmed. When RTA and AdGFP-RTB was used individually, neither was toxic to the cells. When they were applied together, significant cell death was observed in all of the cell lines tested. The cell-killing effect correlated with the amount of RTA protein used, with cell mortality at about 60% at 4.8 lag RTA in combination with AdGFP-RTB at 100 pfu/ceU. No major cell killing was seen when RTA was used in combination with a control adenovirus AdGFP. The treatment of healthy HeLa cells with the virus-flee supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death,suggesting the formation of RTA/RTB complex, and a potential by-stander effect.Conclusion: The new approach was successful in vitro. Further modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its poten-tial in cancer therapy.

  14. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  15. 腺病毒介导的秦川牛固醇调节元件结合蛋白2基因(SREBP2)过表达载体的构建与鉴定%Construction and Identification of Recombinant Adenovirus Vector Specific to Qinchuan Cattle(Bos taurus) Derived Sterol Regulatory Element Binding Protein 2 Gene (SREBP2)

    Institute of Scientific and Technical Information of China (English)

    付常振; 刘扬; 昝林森; 王虹; 成功; 王嘉力

    2013-01-01

    Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol.We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector,and packed and amplified the virus for a high titer,as a antecedent work for the further study of cellular level function of SERBP2 gene.Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA.A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No.NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR).The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid.The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease Pine Ⅰ and subsequently transformed into Escherichia coli B J5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV.And then,the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with Pac Ⅰ and transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV,and to collect virus of high titer.The viral titer of AdSREBP2 and Ad-CMV was 7 × 108 and 1.3 × 109 GFU/mL respectively,measured by green fluorescent protein (GFP) labelled method.Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues.The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h,determined by quantitative Real-time PCR.The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular

  16. Downmodulation of El A Protein Expression as a Novel Strategy to Design Cancer-Selective Adenoviruses

    Directory of Open Access Journals (Sweden)

    Hong Jiang

    2005-08-01

    Full Text Available Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of ElA protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CRi (Delta-39, CR2 (Delta-24 regions, or both regions (Delta-24/39 of the ElA protein. Delta-39, Delta-24 induced a cytopathic effect with similar efficiency in glioma cells, a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, ElA protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CRi mutant ElA proteins, inhibition of the proteasome activity resulted in the striking rescue of ElA levels. We conclude that the level of ElA protein is a critical determinant of oncolytic phenotype, we propose a completely novel strategy for the design, construction of conditionally replicative adenoviruses.

  17. Adenovirus-mediated gene transfer to tumor cells.

    Science.gov (United States)

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  18. A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

    A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

  19. Oncolytic adenovirus-mediated therapy for prostate cancer

    Directory of Open Access Journals (Sweden)

    Sweeney K

    2016-07-01

    future systemic delivery of oncolytic adenoviruses. Keywords: replication selective, virotherapy, combination therapy, clinical trials, gene deletion, transgene

  20. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    Science.gov (United States)

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  1. Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid▿

    OpenAIRE

    Schoehn, Guy; El Bakkouri, Majida; Fabry, Céline M. S.; Billet, Oliver; Leandro F. Estrozi; Le, Van Long; Curiel, David T.; Kajava, Andrey V; Ruigrok, Rob W. H.; Eric J Kremer

    2008-01-01

    There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because AdV-induced disease is relatively species specific, vectors derived from nonhuman serotypes may have wider clinical potential based, in part, on the lack of ubiquitous memory immunity. Whereas a few of the human serotype capsids have been studied at the structural level, none of the nonhuman serotypes has been analyzed. The basis laid by the analysis of human AdV (hAdV) has allowed us to determine ...

  2. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  3. Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the expression of coxsackievirus and adenovirus receptor (CAR) and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.METHODS: Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirusbased gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein (GFP) gene.RESULTS: All the colon cancer cell lines examined (HT29,LS180, SW480, SW948 and SW1116) expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis.Further, all cells we examined were efficiently infected with adenoviral vector-GFP.CONCLUSION: The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.

  4. Reassessing culture media and critical metabolites that affect adenovirus production.

    Science.gov (United States)

    Shen, Chun Fang; Voyer, Robert; Tom, Roseanne; Kamen, Amine

    2010-01-01

    Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.

  5. Oncolytic Adenoviruses for Gynecologic Cancer

    OpenAIRE

    Bauerschmitz, Gerd Johannes

    2007-01-01

    Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly deve...

  6. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  7. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  8. Impact of endogenous Inflammation caused by adenovirus as a vector in gene therapy:experiment with mice models of acute lung injury%以腺病毒为载体的基因治疗对急性肺损伤治疗效果的影响及其意义

    Institute of Scientific and Technical Information of China (English)

    王燕; 陆权; 滕国良; Feistein S.Sheldon; Micheal F.Beers; Aron B.Fisher

    2008-01-01

    Objective To evaluate the impact of endogenous inflammation caused by adenovims as a vector of gene therapy on monse model of acute lung injury(ALI).Methods black C57/B6 mice randomly divided into 4 research groups:(1)adenovirus encoded-lacZ genetreatment group(AdLacZ):n=AdLscZ reagent through intranasal administration to infect mice lungs at 48h before 100%O2 inhalation to induce ALI mouse model,which companied by mice survival rates recorded.B-gal protein activity in lung wag detected to show the level of LacZ DNA transgenic protein activity;meanwhile,the indices of lung wet/dry ratio and bronchial alveolar lavage liquid(BALF)analysis with protein concentration and cell elagsification were detected.Results The method of adenovirus-mediated gene therapy with intranagal administration restthed in IJacZ DNA transgenic protein activity to keep effective highly expression in lung.and the expression level maintained 2.fold increusing even after 72 h of O.inhalation compared to that before O2 inhalation(3.688 U/mg vs 1.589 U/mg);AdLacZ mice had more subjective to O2 inhalation compared to other groups.the 50%mice survival time of this group wag shorter compared to that of the PBS group [(86±3)h vs(94±7)h];also in AdLacZ group,the level of nucleated cell counting in BALF was statistically higher compared to other groups at 48 h of O2 inhalation,which following with 50%-level decreased within anther 24 h O2 inhalation;on the contrary,the level of lung wet/dry ratio and protein concentration in BALF didn,t show remarkably decreasing.Conclusion The endogenous inflammation caused by adenovirus as a vector in ALl gene therapy is temporary and rapidly weaken after getting a peak,however,enough attention still should be paid attention when evaluating the effect of gene therapy.%目的 评价腺病毒自身的致炎作用在以其为载体的基因治疗中,对小鼠急性肺损伤(ALI)治疗效果的影响及意义.方法 C57/B6雄性小鼠随机分为4组:(1)以腺病毒为

  9. siRNA干扰人BMP9重组腺病毒载体的构建及其促进乳腺癌SK-BR-3细胞增殖%Construction of recombinant adenovirus vector interfering the expression of human BMP9 and promoting the proliferation of breast cancer SK-BR-3 cells

    Institute of Scientific and Technical Information of China (English)

    刘月红; 王科; 孙笑笑; 万绍恒; 王维; 陈莹莹; 张彦

    2013-01-01

    目的 筛选特异性干扰人BMP9基因的siRNA序列并制备重组腺病毒AdsiBMP9,探讨RNAi人BMP9基因后对乳腺癌SK-BR-3细胞增殖能力的影响.方法 设计制备3对干扰人BMP9的双链DNA序列,亚克隆至Pses-Hus质粒中获得Pses-Hus-siBMP9质粒,脂质体转染乳腺上皮细胞HBL-100筛选有效干扰质粒,构建重组腺病毒并感染SK-BR-3细胞,RT-PCR,Western blot检测BMP9表达,MTT检测细胞增殖能力.结果 成功构建并筛选出针对人BMP9基因的有效干扰质粒并包装成腺病毒,病毒滴度为1×1010IU/mL,感染SK-BR-3细胞显示BMP9在转录水平和翻译水平表达量显著低于对照组和空白组(P<0.05),第5天AdsiBMP9组细胞的增殖率显著高于AdsiNC组(P<0.05).结论 成功构建特异性沉默人BMP9基因的siRNA腺病毒载体,可有效抑制SK-BR-3细胞中BMP9基因的表达从而促进该细胞增殖.%Objective To screen specific small interfering RNA(siRNA) target human BMP9 gene and to prepare recombinant adenovirus vector AdsiBMP9 for investigation of its effects on the proliferation of breast cancer SK-BR-3 cells. Methods Three pairs of double-stranded DNA fragments for silencing human BMP9 were designed and synthesized, then subcloned into the shuttle plasmid Pses-Hus. The recombinant plasmids Pses-Hus-siBMP9 were transfected into the breast epithelial cells HBL-100 by lipofectamine transfection reagent, screened the effective interfering plasmid, constructed AdsiBMP9 and infected SK-BR-3 cells. The expression level of BMP9 mRNA and protein were detected by RT-PCR and western blot. The proliferation of SK-BR-3 cells were observed with MTT assay. Results The recombinant plasmid Pses-Hus-siBMP9 and recombinant adenovirus AdsiBMP9 were successfully constructed and its titer was 1 × 1010 IU/mL. Compared to the negative and non-infected controls, the expres- sion of BMP9 gene was significantly inhibited after the SK-BR-3 cells were infected by AdsiBMP9. SK-BR-3 cells infected with

  10. Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation%反义VEGF165腺病毒重组体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王家宁; 黄永章; 王俊峰; 王卫民; 李瑞明; 张群林

    2001-01-01

    objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.%目的:构建含人反义血管内皮生长因子165(VEGF165)基因的重组腺病毒载体,为采用反义VEGF165RNA防治肿瘤的研究奠定基础。方法:将人VEGF165 cDNA反向插入到穿梭质粒pHCMVSP1A的CMV启动子之下,即pAd-ahVEGF165。后者与pJM17通过脂质体共转染293细胞,经同源重组获得含人反义VEGF165基因的重组腺病毒Ad-ahVEGF165。通过PCR共扩增法鉴别Ad-ahVEGF165的正确与否。根据260nm的紫外光吸收值计算病毒滴度。结果:VEGF165 cDNA成功地反向插入了pHCMVSP1A载体,以重组病毒基因组DNA

  11. Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control

    DEFF Research Database (Denmark)

    Sorensen, Maria R; Holst, Peter J; Pircher, Hanspeter

    2009-01-01

    Antigen-specific immunotherapy is an attractive strategy for cancer control. In the context of antiviral vaccines, adenoviral vectors have emerged as a favorable means for immunization. Therefore, we chose a strategy combining use of these vectors with another successful approach, namely linkage...... of the vaccine antigen to invariant chain (Ii). To evaluate this strategy we used a mouse model, in which an immunodominant epitope (GP33) of the LCMV glycoprotein (GP) represents the tumor-associated neoantigen. Prophylactic vaccination of C57BL/6 mice with a replication-deficient human adenovirus 5 vector...... the tumor degradation. Finally, Ad-Ii-GP but not Ad-GP vaccination can break the immunological non-reactivity in GP transgenic mice indicating that our vaccine strategy will prove efficient also against endogenous tumor antigens....

  12. Construction of recombinant adenovirus vector for human matrix metalloproteinase-1 gene and detection of collagen type III degradation in vitro%构建人基质金属蛋白酶1基因重组腺病毒载体及体外降解Ⅲ型胶原的检测

    Institute of Scientific and Technical Information of China (English)

    杜超; 蒋明德; 曾维政; 郑淑梅

    2014-01-01

    BACKGROUND:Matrix metaloproteinase-1 can degrade extracelular matrix, which is mainly colagen type I, and has the potential to reverse fibrosis tissue. OBJECTIVE:To construct the recombinant adenovirus vector containing human matrix metaloproteinase-1 (hMMP-1) gene with GatewayTM Clone Technology, and observe the capacity of degrading colagen type IIIin vitro. METHODS: The gene hMMP-1 was amplified by using PCR from the pcDNA3.1 plasmid and was cut down by the double endonuclease. The linear gene fragment was connected to the entry vector pENTERTM 1A. Then the entry clone and the destination vectors pJTI™ R4 Dest CMV-N-EmGFP pA Vector recombined using the LR reaction to form the expression clone pAd-hMMP-1-eGFP. The linear pAd-hMMP-1-eGFP cut down by endonucleasePac I was transfected into HEK293A cels to packaging the Ad-hMMP-1-eGFP. The transfected situation was observed under a fluorescence microscope, the target protein expression was detected by western-blot assay and RT-PCR. Cels can be divided into three groups: blank control group: HEK293A cels, AD-EGFP group: HEK293A cels were infected by Ad-eGFP, AD-HMMP1-EGF group: HEK293A cels were infected by Ad-hMMP1-eGFP and colagen type III. The content of colagen type III was detected by ELISA kits after 24, 48 and 72 hours. RESULTS AND CONCLUSION: It was confirmed that the entry vector and the destination vector both contained hMMP-1 target gene by restriction analysis and sequencing. The green fluorescent protein was observed in the 293A cels transfected by the Ad-hMMP-1-eGFP at 4 days. The fluorescence intensity was the highest at 10 days. The virus was colected at 12 days, the viral titer was determined as 4.84 × 1010 PFU/mL, the target protein was efficient expressionvia western-blot assay. Blank control group and AD-EGFP group had no obvious change of colagen content with the extension of time. The rate of colagen degradation in AD-HMMP1-EGFP group was 24%, 56% and 81% respectively at 24, 48, 72 hours. AD

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

    Science.gov (United States)

    Freimuth, Paul I.

    2010-04-06

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

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

    Directory of Open Access Journals (Sweden)

    Johan Rebetz

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

  15. High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Fei Zhong; Zhen-Yu Zhong; Shuang Liang; Xiu-Jin Li

    2006-01-01

    AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1~1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

  16. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  17. 鼠胰岛素样生长因子-1重组腺病毒的构建及其在胰岛β细胞的表达%Construction of Recombinant Adenovirus Vector Containing Rat Insulin-like Growth Factor 1 Gene and Its Expression in Islet β-cells

    Institute of Scientific and Technical Information of China (English)

    陈志红; 李堂; 胡素娟; 王桂琴

    2009-01-01

    This study was aimed to construct the recombinant adenovirus containing rat insulin-like growth factor 1(rIGF-1),and then to investigate its expression in islet β-cells. RNA was extracted using Trizol from rat livers. rIGF-1 cDNA was obtained using RT-PCR. The purified RT-PCR products and pAdTrack-CMV were digested using Bgl II and EcoR V and religated by T4 DNA ligase,then transformed into electro-competent JM109 bacteria and selected on Kanamycin LB plates. This plasmid pAd-CMV-rIGF-1 was linearized by PmeI and co-transformed into electro-competent BJ5183 bacteria with pAdEasy-1 and selected on Kanamycin LB plates. After having been screened,the extracted plasmid of positive bacteria was transfected into HEK 293 cells with liposome and was identified by the green fluorescence protein (GFP) expression. The recombinant adenovirus encoding rIGF-1 was named Ad-rIGF-1,and the viral particles were further amplified,purified,and its titer was about 4.0×10~8pfu/ml. Ad-rIGF-1 was transfected into rat pancreatic β cell lines-RINm5F cells,RT-PCR was carried out to detect the transfer genes. rIGF-1 protein in cells culture supernatants was detected by ELISA method,and its concentration was 91.6±26.8ng/ml. rIGF-1 was present in Ad-rIGF-1-infected RINm5F cells as measured by Western blotting. The recombinant adenovirus vector containing rIGF-1 was constructed successfully,and the rIGF-1 protein was expressed by RINm5F cells. This method provided the mechanism of rIGF-1 to prevent β cell from impairmentand to treat the case of type 1 diabetes.%构建含有鼠胰岛素样生长因子1(rIGF-1)重组腺病毒,研究其在胰岛β细胞的表达.Trizol 一步法提取大鼠肝脏组织总RNA,RT-PCR法合成鼠胰岛素样生长因1(rIGF-1) cDNA;PCR纯化产物和pAdTrack-CMV分别经BglⅡ和EcoRⅤ双酶切后纯化,T4 DNA 连接酶连接,转化JM109感受态细菌,卡那霉素平板筛选阳性克隆扩增培养,抽提质粒;经BglⅡ和EcoRⅤ双酶切及测序鉴定后,

  18. Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

    Science.gov (United States)

    Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

    2016-02-01

    The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

  19. SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

    Science.gov (United States)

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin; Mund, Andreas; Wimmer, Peter; Schubert, Tobias; Groitl, Peter; Will, Hans; Dobner, Thomas

    2013-01-01

    Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming

  20. An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy

    Institute of Scientific and Technical Information of China (English)

    ZI LAI ZHANG; WEI GUO ZOU; CHUN XIA LUO; BING HUA LI; JIN HUI WANG; LAN YING SUN; QI JUN QIAN; XIN YUAN LIU

    2003-01-01

    ONYXONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them.To date,clinicaltrials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone.In this paper,we put forward a novel concept of Gene-Viro Therapy strategy and in this way,we constructed an armed therapeutic onco1ytic adenovirus system,ZD55-gene,whichis not only deleted of E1B 55-kD gene similar to ONYX-015,but also armed with foreign antitumor gene.ZD55-gene exhibited similar cytopathic effects and replication Kinetics to that of ONYX-015 in vitro.Importantly,the carried gene 1s expressed and the expression level can increase with the replication of virus.Consequently,a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer.Our data demonstratedthat ZD55-gene,which utilizingthe Gene-ViroTherapy strategy,is more efficacious than each individual component in vivo.

  1. Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Qi; CHEN Dayu; FU Xiangning; ZU Yukun

    2006-01-01

    The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000 μg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

  2. Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR.

    Science.gov (United States)

    Herbert, Rebecca; Baron, Jana; Batten, Carrie; Baron, Michael; Taylor, Geraldine

    2014-02-26

    Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induc