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Sample records for adenovirus ad vector

  1. Alternative Serotype Adenovirus Vaccine Vectors Elicit Memory T Cells with Enhanced Anamnestic Capacity Compared to Ad5 Vectors

    PubMed Central

    Penaloza-MacMaster, Pablo; Provine, Nicholas M.; Ra, Joshua; Borducchi, Erica N.; McNally, Anna; Simmons, Nathaniel L.; Iampietro, Mark J.

    2013-01-01

    The failure of the adenovirus serotype 5 (Ad5) vector-based human immunodeficiency virus type 1 (HIV-1) vaccine in the STEP study has led to the development of adenovirus vectors derived from alternative serotypes, such as Ad26, Ad35, and Ad48. We have recently demonstrated that vaccines using alternative-serotype Ad vectors confer partial protection against stringent simian immunodeficiency virus (SIV) challenges in rhesus monkeys. However, phenotypic differences between the T cell responses elicited by Ad5 and those of alternative-serotype Ad vectors remain unexplored. Here, we report the magnitude, phenotype, functionality, and recall capacity of memory T cell responses elicited in mice by Ad5, Ad26, Ad35, and Ad48 vectors expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP). Our data demonstrate that memory T cells elicited by Ad5 vectors were high in magnitude but exhibited functional exhaustion and decreased anamnestic potential following secondary antigen challenge compared to Ad26, Ad35, and Ad48 vectors. These data suggest that vaccination with alternative-serotype Ad vectors offers substantial immunological advantages over Ad5 vectors, in addition to circumventing high baseline Ad5-specific neutralizing antibody titers. PMID:23152535

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

    PubMed

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

    2009-10-01

    The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon-containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5. PMID:19603000

  3. Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors

    PubMed Central

    Teigler, Jeffrey E.; Penaloza-MacMaster, Pablo; Obeng, Rebecca; Provine, Nicholas M.; Larocca, Rafael A.; Borducchi, Erica N.

    2014-01-01

    Hexon modification of adenovirus type 5 (Ad5) vectors with the hypervariable regions (HVRs) of Ad48 has been shown to allow Ad5HVR48 vectors to circumvent the majority of the preexisting Ad5-neutralizing antibodies. However, it remains unclear whether modifying hexon HVRs impacts innate or adaptive immune responses elicited by this vector. In this study, we investigated the influence of the HVR substitution of Ad5 on innate and adaptive immune responses following vaccination. Ad5HVR48 displayed an intermediate level of innate immune cytokines and chemokines relative to those of Ad5 and Ad48, consistent with its chimeric nature. Hepatotoxicity was observed after Ad5 immunization but not after Ad5HVR48 or Ad48 immunization. However, the CD8+ T-cell responses elicited by Ad5HVR48 vectors displayed a partially exhausted phenotype, as evidenced by the sustained expression of programmed death 1 (PD-1), decreased effector-to-central memory conversion, and reduced memory recall responses, similar to those elicited by Ad5 vectors and in contrast to those induced by Ad48 vectors. Taken together, these results indicate that although Ad5HVR48 largely bypasses preexisting Ad5 neutralizing antibodies and shows reduced hepatotoxicity compared to that of Ad5, it induces adaptive immune phenotypes that are functionally exhausted similar to those elicited by Ad5. PMID:24943382

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

    PubMed Central

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

    2015-01-01

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

  5. Clinical Assessment of a Novel Recombinant Simian Adenovirus ChAdOx1 as a Vectored Vaccine Expressing Conserved Influenza A Antigens

    PubMed Central

    Antrobus, Richard D; Coughlan, Lynda; Berthoud, Tamara K; Dicks, Matthew D; Hill, Adrian VS; Lambe, Teresa; Gilbert, Sarah C

    2014-01-01

    Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection. PMID:24374965

  6. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens.

    PubMed

    Antrobus, Richard D; Coughlan, Lynda; Berthoud, Tamara K; Dicks, Matthew D; Hill, Adrian Vs; Lambe, Teresa; Gilbert, Sarah C

    2014-03-01

    Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection. PMID:24374965

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

    PubMed

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

    2013-01-01

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

  8. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

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

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies. PMID:27076309

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    DOE PAGESBeta

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; Borducchi, Erica N.; Iampietro, M. Justin; Bricault, Christine A.; Teigler, Jeffrey E.; Blackmore, Stephen; Parenteau, Lily; Wagh, Kshitij; et al

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved tomore » have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.« less

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

    SciTech Connect

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

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

  12. Adenovirus vectors targeting distinct cell types in the retina.

    PubMed

    Sweigard, J Harry; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-04-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5DeltaRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5DeltaRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5DeltaRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5DeltaRGD vectors. PMID:19892875

  13. Adenovirus Dodecahedron, as a Drug Delivery Vector

    PubMed Central

    Zochowska, Monika; Paca, Agnieszka; Schoehn, Guy; Andrieu, Jean-Pierre; Chroboczek, Jadwiga; Dublet, Bernard; Szolajska, Ewa

    2009-01-01

    Background Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. Principal Findings Dodecahedron (Dd) structure is preserved at up to about 50°C at pH 7–8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37°C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. Conclusions/Significance Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs. PMID:19440379

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Weaver, Eric A; Barry, Michael A

    2013-01-01

    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. PMID:23991187

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

    Gao, Dong-Sheng; Li, Xiao-Jing; Wan, Wen-Yan; Li, Hong-Jie; Wang, Xiao-Xue; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry. PMID:26850542

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

    PubMed

    Zhang, Zheng; Wang, Guoxian; Li, Chen; Liu, Danping

    2013-08-01

    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(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

  20. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single dose in ovo vaccination with a replication competent adenovirus (RCA) -free human adenovirus vector (Ad5) encoding an avian AI virus H5 hemagglutinin. Vaccinated chickens were protected against both H5N1 and H5...

  1. A CD46-binding chimpanzee adenovirus vector as a vaccine carrier.

    PubMed

    Tatsis, Nia; Blejer, Ariella; Lasaro, Marcio O; Hensley, Scott E; Cun, Ann; Tesema, Lello; Li, Yan; Gao, Guang-Ping; Xiang, Zhi Q; Zhou, Dongming; Wilson, James M; Ertl, Hildegund C J

    2007-03-01

    A replication-defective chimeric vector based on the chimpanzee adenovirus serotype C1 was developed and tested as a vaccine carrier in mice. The AdC1 virus is closely related to human adenoviruses of subgroup B2 and uses CD46 for cell attachment. To overcome poor growth of E1-deleted AdC1 vectors on cell lines that provide the E1 of adenovirus of the human serotype 5 (AdHu5) virus in trans, the inverted terminal repeats and some of the early genes of AdC1 were replaced with those from AdC5, a chimpanzee origin adenovirus of subfamily E. The chimeric AdC1/C5 vector efficiently transduces CD46-expressing mouse dendritic cells (DCs) in vitro and initiates their maturation. Transduction of DCs in vivo is inefficient in CD46 transgenic mice. The AdC1/C5 vector induces transgene product-specific B- and CD8(+) T-cell responses in mice. Responses are slightly higher in wild-type mice than in CD46 transgenic mice. Transgene product-specific T-cell responses elicited by the AdC1/C5 vector can be increased by priming or boosting with a heterologous adenovirus vector. Pre-existing immunity to adenovirus of the common human serotype 5 does not affect induction of cell-mediated immune responses by the AdC1/C5 vector. This vector provides an additional tool in a repertoire of adenovirus-based vaccine vectors. PMID:17228314

  2. Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection

    PubMed Central

    Tamminga, Cindy; Sedegah, Martha; Maiolatesi, Santina; Fedders, Charlotte; Reyes, Sharina; Reyes, Anatalio; Vasquez, Carlos; Alcorta, Yolanda; Chuang, Ilin; Spring, Michele; Kavanaugh, Michael; Ganeshan, Harini; Huang, Jun; Belmonte, Maria; Abot, Esteban; Belmonte, Arnel; Banania, JoGlenna; Farooq, Fouzia; Murphy, Jittawadee; Komisar, Jack; Richie, Nancy O; Bennett, Jason; Limbach, Keith; Patterson, Noelle B; Bruder, Joseph T; Shi, Meng; Miller, Edward; Dutta, Sheetij; Diggs, Carter; Soisson, Lorraine A; Hollingdale, Michael R; Epstein, Judith E; Richie, Thomas L

    2013-01-01

    Background: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. Methodology/Principal Findings: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 1010 particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range < 50–1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2–38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38–2550) and for AMA1 of 1303 (range 435–4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively. Significance: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015. PMID:23899517

  3. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

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

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8+ T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides ‘self-adjuvanting’ activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  4. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

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

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  5. Efficient construction of recombinant adenovirus expression vector of the Qinchuan cattle LYRM1 gene.

    PubMed

    Li, Y K; Fu, C Z; Zhang, Y R; Zan, L S

    2015-01-01

    In this study, we cloned the coding DNA sequence (CDS) region of Qinchuan cattle LYR motif-containing 1 (LYRM1) and constructed a recombinant adenovirus expression vector to examine the function of LYRM1 on the cellular level. Total RNA was extracted from the adipose tissue of Qinchuan cattle, cDNA was obtained by reverse transcription, and polymerase chain reaction was used to amplify the CDS region of the LYRM1 gene. The CDS-containing fragment was inserted into the shuttle vector pAdTrack-CMV to construct pAdTrack-CMV-LYRM1 vector. After linearization of pAdTrack-CMV-LYRM1 and the negative control vector pAdTrack-CMV by restriction endonuclease PmeI, the vectors were transformed into Escherichia coli BJ5183 containing pAdEasy-1 to obtain the recombinant adenovirus vector pAd-LYRM1 and pAd-CMV through homologous recombination. pAd-LYRM1 and pAd-CMV were then digested by PacI and transfected into the 293A cell line. The recombinant adenovirus Ad-LYRM1 and Ad-CMV was obtained at a concentration of 7 x 108 and 1.3 x 109 green fluorescent units/mL, respectively. Preadipocytes derived from Qinchuan cattle were separately infected with Ad-LYRM1 and Ad- CMV. Quantitative real-time polymerase chain reaction demonstrated that the expression of LYRM1 was increased by approximate 28,000-folds after the infection with recombinant adenovirus for 48 h. In conclusion, we successfully cloned the CDS region of the Qinchuan cattle LYRM1 gene, constructed the recombinant adenovirus expression vector, and obtained the adenovirus with high titer, providing valuable materials for studying the function of LYRM1 at the cellular level. PMID:26345880

  6. Chimpanzee Adenovirus Vector Ebola Vaccine - Preliminary Report.

    PubMed

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

    2014-11-26

    Background The unprecedented 2014 epidemic of Ebola virus disease (EVD) has prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. Methods We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10(10) particle units or 2×10(11) particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 4 weeks after vaccination. Results In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10(11) particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10(11) particle-unit dose than in the group that received the 2×10(10) particle-unit dose (geometric mean titer against the Zaire antigen, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2x10(11) particle-unit dose than among those who received the 2×10(10) particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07). Conclusions Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×10(11) particle-unit dose, glycoprotein Zaire-specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates. Clinical trials

  7. NF-κB promotes leaky expression of adenovirus genes in a replication-incompetent adenovirus vector

    PubMed Central

    Machitani, M.; Sakurai, F.; Wakabayashi, K.; Nakatani, K.; Shimizu, K.; Tachibana, M.; Mizuguchi, H.

    2016-01-01

    The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes. PMID:26814140

  8. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag–pol–nef antigen

    PubMed Central

    Herath, S.; Le Heron, A.; Colloca, S.; Bergin, P.; Patterson, S.; Weber, J.; Tatoud, R.; Dickson, G.

    2015-01-01

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  9. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag-pol-nef antigen.

    PubMed

    Herath, S; Le Heron, A; Colloca, S; Bergin, P; Patterson, S; Weber, J; Tatoud, R; Dickson, G

    2015-12-16

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  10. Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

    PubMed Central

    Wold, William S.M.; Toth, Karoly

    2015-01-01

    Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through the natural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competent adenovirus vectors are safe and have therapeutic activity. PMID:24279313

  11. Development and assessment of human adenovirus type 11 as a gene transfer vector.

    PubMed

    Stone, Daniel; Ni, Shaoheng; Li, Zong-Yi; Gaggar, Anuj; DiPaolo, Nelson; Feng, Qinghua; Sandig, Volker; Lieber, André

    2005-04-01

    Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism. PMID:15795294

  12. Human Adenovirus Serotype 3 Vector Packaged by a Rare Serotype 14 Hexon

    PubMed Central

    Ma, Qiang; Liu, Qian; Lu, Xiaomei; Zhou, Rong

    2016-01-01

    Recombinant adenovirus serotype 3 (rAd3), which infects cells through the receptor desmoglein 2 (DSG2), has been investigated as a vector for gene therapy or vaccination. However, pre-existing anti-vector immunity may limit the practical application of rAd3. In this study, we investigated the seroprevalence and neutralizing antibody (NAb) titers to Ad3 and alternate serotypes in normal healthy adults in southern China. Sera samples had a high seroprevalence (80.00%) against Ad3 and Ad7 (85.83%), compared with Ad14 (22.50%). Furthermore, 19.17% and 25.83% of samples had high-titer neutralizing antibodies to Ad3 and Ad7, respectively, compared with 3.33% against Ad14. We constructed a chimeric adenovirus, rAd3H14, designed to evade anti-vector immunity by replacing the enhanced green fluorescent protein (EGFP)-expressing hexon of the rAd3EGFP vector with a hexon from Ad14. The chimeric vector rAd3H14 was not neutralized in vitro efficiently by Ad3 NAbs using sera from mice and normal healthy human volunteers. Furthermore, in contrast to the unmodified vector rAd3EGFP, rAd3H14 induced robust antibody responses against EGFP in mice with high levels of pre-existing anti-Ad3 immunity. In conclusion, the chimeric vector rAd3H14 may be a useful alternative vector in adult populations with a high prevalence of Ad3 NAbs. PMID:27328032

  13. Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

    PubMed

    Mizuguchi, Hiroyuki; Hayakawa, Takao

    2002-03-01

    There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments. PMID:11896439

  14. Construction of an adenovirus type 7a E1A- vector.

    PubMed Central

    Abrahamsen, K; Kong, H L; Mastrangeli, A; Brough, D; Lizonova, A; Crystal, R G; Falck-Pedersen, E

    1997-01-01

    A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector. PMID:9343264

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

    PubMed Central

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

    2015-01-01

    Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors. PMID:26376928

  16. Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses.

    PubMed

    Mück-Häusl, Martin; Solanki, Manish; Zhang, Wenli; Ruzsics, Zsolt; Ehrhardt, Anja

    2015-04-30

    Recombinant adenoviruses containing a double-stranded DNA genome of 26-45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies. PMID:25609697

  17. A chimeric adenovirus with an Ad 3 fiber knob modification augments glioma virotherapy

    PubMed Central

    Nandi, Suvobroto; Ulasov, Ilya V.; Rolle, Cleo E.; Han, Yu; Lesniak, Maciej S.

    2009-01-01

    Background Malignant gliomas remain refractory to treatment despite advances in chemotherapy and surgical techniques. Viral vectors developed to treat gliomas have had low transduction capabilities, limiting their use. Gliomas over-express CD46, CD80, and CD86, all of which bind adenovirus serotype 3. Methods To increase the infectivity and replication of oncolytic vectors in malignant brain tumors, we created a conditionally replicating adenovirus, CRAd-Survivin-5/3, which contains a survivin promoter-driving E1A and a chimeric fiber consisting of adenovirus serotype 3 knob. Results In vitro, this modified CRAd showed ten- to 100-fold increased cytotoxicity against glioma cells. Ex vivo analysis of primary glioblastoma multiforme samples infected with CRAd-Survivin-5/3 showed an increase in cytotoxicity of 20–30% compared to adenovirus wild-type (AdWT). Innormal human astrocytes and normal brain tissues, CRAd-Survivin-5/3 exhibited 30–40% and 10–15% lower cytotoxicity than AdWT, respectively. In an intracranial xenograft model of glioma, this oncolytic virus increased tumor-free survival and overall lifespan by 50% compared to controls (p < 0.05). Conclusions CRAd-Survivin-5/3 represents an attractive alternative to existing vectors and should be tested further in the pre-clinical setting. PMID:19688792

  18. Strategies to overcome host immunity to adenovirus vectors in vaccine development

    PubMed Central

    Thacker, Erin E; Timares, Laura; Matthews, Qiana L

    2013-01-01

    The first clinical evaluations of adenovirus (Ad)-based vectors for gene therapy were initiated in the mid-1990s and led to great anticipation for future utility. However, excitement surrounding gene therapy, particularly Ad-based therapy, was diminished upon the death of Jesse Gelsinger, and recent discouraging results from the HIV vaccine STEP trial have brought efficacy and safety issues to the forefront again. Even so, Ad vectors are still considered among the safest and most effective vaccine vectors. Innate and pre-existing immunity to Ad mediate much of the acute toxicities and reduced therapeutic efficacies observed following vaccination with this vector. Thus, innovative strategies must continue to be developed to reduce Ad-specific antigenicity and immune recognition. This review provides an overview and critique of the most promising strategies, including results from preclinical trials in mice and nonhuman primates, which aim to revive the future of Ad-based vaccines. PMID:19485756

  19. Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production.

    PubMed

    Koizumi, Naoya; Yamaguchi, Tomoko; Kawabata, Kenji; Sakurai, Fuminori; Sasaki, Tomomi; Watanabe, Yoshiteru; Hayakawa, Takao; Mizuguchi, Hiroyuki

    2007-02-01

    Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses. PMID:17237426

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

    PubMed

    Shimizu, Kahori; Sakurai, Fuminori; Tomita, Kyoko; Nagamoto, Yasuhito; Nakamura, Shin-Ichiro; Katayama, Kazufumi; Tachibana, Masashi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2014-01-01

    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. PMID:26015975

  1. Rapid generation of fowl adenovirus 9 vectors.

    PubMed

    Pei, Yanlong; Griffin, Bryan; de Jong, Jondavid; Krell, Peter J; Nagy, Éva

    2015-10-01

    Fowl adenoviruses (FAdV) have the largest genomes of any fully sequenced adenovirus genome, and are widely considered as excellent platforms for vaccine development and gene therapy. As such, there is a strong need for stream-lined protocols/strategies for the generation of recombinant adenovirus genomes. Current genome engineering strategies rely upon plasmid based homologous recombination in Escherichia coli BJ5183. This process is time-consuming, involves multiple cloning steps, and low efficiency recombination. This report describes a novel system for the more rapid generation of recombinant fowl adenovirus genomes using the lambda Red recombinase system in E. coli DH10B. In this strategy, PCR based amplicons with around 50 nt long homologous arms, a unique SwaI site and a chloramphenicol resistance gene fragment (CAT cassette), are introduced into the FAdV-9 genome in a highly efficient and site-specific manner. To demonstrate the efficacy of this system we generated FAdV-9 ORF2, and FAdV-9 ORF11 deleted, CAT marked and unmarked FAdV-9 infectious clones (FAdmids), and replaced either ORF2 or ORF11, with an EGFP expression cassette or replaced ORF2 with an EGFP coding sequence via the unique SwaI sites, in approximately one month. All recombinant FAdmids expressed EGFP and were fully infectious in CH-SAH cells. PMID:26238923

  2. Mutation in fiber of adenovirus serotype 5 gene therapy vector decreases liver tropism

    PubMed Central

    Wang, Zhen; Wang, Baoming; Lou, Junfang; Yan, Jingyi; Gao, Lei; Geng, Ranshen; Yu, Bin

    2014-01-01

    Recombinant adenovirus (Ad) vectors are widely used for both in vitro and in vivo gene transfer. However, intravenous administration of Ad vectors results mainly in hepatocyte transduction and subsequent hepatotoxicity. Coxsackie-adenovirus receptor (CAR) and αvβ integrins, which are functional receptors for the fiber and penton proteins, respectively, are the tropism determinants of Ad type 5 (Ad5). We previously developed a system for rapid construction of fiber-modified Ad5 vectors. We also constructed a fiber-modified Ad5 containing an Arg-Gly-Asp (RGD) motif in the HI-loop and showed that it could enhance anti-tumor effects in vitro and in vivo. Here, we constructed a novel Ad5 vector containing two amino acid mutations in the AB loop of the fiber-modified Ad5 fiber knob and showed that it could significantly reduce liver tropism and increase gene transfer in low-CAR or CAR-deficient cancer cells following intravascular delivery. However, anti-tumor effects of the fiber-mutated Ad5 expressing HSV-TK under control of the hTERT promoter was not found when compared with an unmodified Ad5 vector in cancer lines expressing different levels of CAR, likely due to the activity of the hTERT promoter being lower than that of the CMV promoter. Nevertheless, this study describes an enhanced Ad5 vector for intravascular gene delivery, and further modifications such as changes in the promoter may facilitate the development of this vector for cancer treatment. PMID:25663991

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

    PubMed

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

    2013-01-01

    Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo. PMID:24326926

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    1997-01-01

    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. PMID:9343173

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

    PubMed

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-01

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. PMID:27176913

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

    PubMed Central

    2013-01-01

    Background Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. Methods Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. Results A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. Conclusions Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials. PMID:24304565

  8. A porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an H5N1 virus disease model.

    PubMed

    Patel, Ami; Tikoo, Suresh; Kobinger, Gary

    2010-01-01

    Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5. PMID:21179494

  9. Adenovirus vector-induced immune responses in nonhuman primates: responses to prime boost regimens1

    PubMed Central

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

    2009-01-01

    In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with pre-existing neutralizing antibodies against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee derived Ad (AdC) vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the AdC vectors induced higher T and B cell responses than repeated immunizations with the AdHu5 vector especially in AdHu5-pre-exposed macaques. PMID:19414814

  10. Expression of an engineered soluble coxsackievirus and adenovirus receptor by a dimeric AAV9 vector inhibits adenovirus infection in mice.

    PubMed

    Röger, C; Pozzuto, T; Klopfleisch, R; Kurreck, J; Pinkert, S; Fechner, H

    2015-06-01

    Immunosuppressed (IS) patients, such as recipients of hematopoietic stem cell transplantation, occasionally develop severe and fatal adenovirus (Ad) infections. Here, we analyzed the potential of a virus receptor trap based on a soluble coxsackievirus and Ad receptor (sCAR) for inhibition of Ad infection. In vitro, a dimeric fusion protein, sCAR-Fc, consisting of the extracellular domain of CAR and the Fc portion of human IgG1 and a monomeric sCAR lacking the Fc domain, were expressed in cell culture. More sCAR was secreted into the cell culture supernatant than sCAR-Fc, but it had lower Ad neutralization activity than sCAR-Fc. Further investigations showed that sCAR-Fc reduced the Ad infection by a 100-fold and Ad-induced cytotoxicity by ~20-fold. Not only was Ad infection inhibited by sCAR-Fc applied prior to infection, it also inhibited infection when used to treat ongoing Ad infection. In vivo, sCAR-Fc was delivered to IS mice by an AAV9 vector, resulting in persistent and high (>40 μg ml(-1)) sCAR-Fc serum levels. The sCAR-Fc serum concentration was sufficient to significantly inhibit hepatic and cardiac wild-type Ad5 infection. Treatment with sCAR-Fc did not induce side effects. Thus, sCAR-Fc virus receptor trap may be a promising novel therapeutic for treatment of Ad infections. PMID:25786873

  11. [Development and Characterization of a Novel Adenovirus Vector Exhibiting MicroRNA-mediated Suppression of the Leaky Expression of Adenovirus Genes].

    PubMed

    Shimizu, Kahori

    2015-01-01

    Replication-incompetent adenovirus (Ad) vectors have gained attention as gene delivery vehicles. Theoretically, no Ad genes should be expressed following transduction; however, Ad genes are expressed from the vector genome, leading to induction of cellular immunity against Ad proteins and Ad protein-induced toxicity. To suppress the leaky expression of Ad genes, a microRNA (miRNA)-regulated gene expression system was utilized. We developed novel Ad vectors by incorporating targeted sequences of miR-122a or miR-142-3p, which exhibit liver- or spleen-specific expression, respectively, in the 3'-untranslated region (UTR) of the E2A, E4, or pIX genes. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional human embryonic kidney 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold in an miRNA-dependent manner, compared with a conventional Ad vector, by the insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene (Ad-E4-122aT) expressed 1.5- to 34-fold 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 compared with a conventional Ad vector. miR-122a-mediated suppression of E4 gene expression in the liver significantly reduced the hepatotoxicity that an Ad vector causes via both adaptive and non-adaptive immune responses. Ad-E4-122aT would be a promising framework for efficient gene delivery due to its ability to mediate higher and longer-term transgene expression and lower hepatotoxicity than a conventional Ad vector. PMID:26632150

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors.

    PubMed

    Solanki, Manish; Zhang, Wenli; Jing, Liu; Ehrhardt, Anja

    2016-01-01

    Commonly used human adenovirus (HAdV)-5-based vectors are restricted by their tropism and pre-existing immunity. Here, we characterized novel HAdV-5 vectors pseudotyped with hypervariable regions (HVRs) and surface domains (SDs) of other HAdV types. Hexon-modified HAdV-5 vectors (HV-HVR5, HV-HVR12, HV-SD12 and HV-SD4) could be reconstituted and amplified in human embryonic kidney cells. After infection of various cell lines, we measured transgene expression levels by performing luciferase reporter assays or coagulation factor IX (FIX) ELISA. Dose-dependent studies revealed that luciferase expression levels were comparable for HV-HVR5, HV-SD12 and HV-SD4, whereas HV-HVR12 expression levels were significantly lower. Vector genome copy numbers (VCNs) from genomic DNA and nuclear extracts were then determined by quantitative real-time PCR. Surprisingly, determination of cell- and nuclear fraction-associated VCNs revealed increased VCNs for HV-HVR12 compared with HV-SD12 and HV-HVR5. Increased nuclear fraction-associated HV-HVR12 DNA molecules and decreased transgene expression levels were independent of the cell line used, and we observed the same effect for a hexon-modified high-capacity adenoviral vector encoding canine FIX. In conclusion, studying hexon-modified adenoviruses in vitro demonstrated that HVRs but also flanking hexon regions influence uptake and transgene expression of adenoviral vectors. PMID:26519158

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

    PubMed

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

    2009-05-15

    In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with preexisting neutralizing Abs against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here, we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee-derived Ad vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the chimpanzee-derived Ad vectors induced higher T and B cell responses than did repeated immunizations with the AdHu5 vector, especially in AdHu5-preexposed macaques. PMID:19414814

  16. An Adenovirus Vector with Genetically Modified Fibers Demonstrates Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus Receptor-Independent Cell Entry Mechanism

    PubMed Central

    Dmitriev, Igor; Krasnykh, Victor; Miller, C. Ryan; Wang, Minghui; Kashentseva, Elena; Mikheeva, Galina; Belousova, Natalya; Curiel, David T.

    1998-01-01

    Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor. PMID:9811704

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector

    PubMed Central

    Shayakhmetov, Dmitry M.; Papayannopoulou, Thalia; Stamatoyannopoulos, George; Lieber, André

    2000-01-01

    Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and αv integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34+ cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34+ cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an αv integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34+ cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34+ cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34+ cells expressing αv integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34+ cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34+ cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34+ c-Kit+ cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34+ c-Kit+ cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells. PMID:10684271

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

    PubMed Central

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

    2015-01-01

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

  20. Immune Recognition of Gene Transfer Vectors: Focus on Adenovirus as a Paradigm

    PubMed Central

    Aldhamen, Yasser Ali; Seregin, Sergey S.; Amalfitano, Andrea

    2011-01-01

    Recombinant Adenovirus (Ad) based vectors have been utilized extensively as a gene transfer platform in multiple pre-clinical and clinical applications. These applications are numerous, and inclusive of both gene therapy and vaccine based approaches to human or animal diseases. The widespread utilization of these vectors in both animal models, as well as numerous human clinical trials (Ad-based vectors surpass all other gene transfer vectors relative to numbers of patients treated, as well as number of clinical trials overall), has shed light on how this virus vector interacts with both the innate and adaptive immune systems. The ability to generate and administer large amounts of this vector likely contributes not only to their ability to allow for highly efficient gene transfer, but also their elicitation of host immune responses to the vector and/or the transgene the vector expresses in vivo. These facts, coupled with utilization of several models that allow for full detection of these responses has predicted several observations made in human trials, an important point as lack of similar capabilities by other vector systems may prevent detection of such responses until only after human trials are initiated. Finally, induction of innate or adaptive immune responses by Ad vectors may be detrimental in one setting (i.e., gene therapy) and be entirely beneficial in another (i.e., prophylactic or therapeutic vaccine based applications). Herein, we review the current understanding of innate and adaptive immune responses to Ad vectors, as well some recent advances that attempt to capitalize on this understanding so as to further broaden the safe and efficient use of Ad-based gene transfer therapies in general. PMID:22566830

  1. Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus.

    PubMed

    Zhou, Dongming; Wu, Te-Lang; Emmer, Kristel L; Kurupati, Raj; Tuyishime, Steven; Li, Yan; Giles-Davis, Wynetta; Zhou, Xiangyang; Xiang, Zhiquan; Liu, Qin; Ratcliffe, Sarah J; Ertl, Hildegund C J

    2013-03-01

    To determine if an ordered and repetitive display of an epitope promoted induction of superior antibody responses, we compared B-cell responses to an influenza A virus epitope that was either encoded as a transgene by an adenovirus (Ad) vector or expressed on the vector's surface. To this end, we constructed a panel of influenza A virus vaccines based on chimpanzee-derived replication-defective adenovirus (AdC) vectors of serotype SAd-V25 also called AdC68. AdC68 vectors were modified to express a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable regions 1 (VR1) or 4 (VR4) of the adenovirus hexon. Additional vectors with wild-type or M2e-modified hexon encoded M2e fused to the influenza A virus nucleoprotein (NP) as a transgene product. Hexon-modified vectors were tested for immunogenicity and efficacy in mice in comparison to vectors with native hexon expressing the M2e-NP fusion protein. Upon priming, vectors expressing M2e within VR1 of hexon induced M2e-specific antibody responses of higher magnitude and avidity than those carrying M2e within VR4 or vectors expressing the M2e as part of a transgene product. CD8(+) T-cell responses to the transgenic NP were comparable between vectors. M2e-specific antibody responses could be boosted by a second dose of the VR1 hexon-modified vector but not by repeated immunization with the VR4 hexon-modified vector. PMID:23229092

  2. Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

    PubMed

    Fernandes, P; Simão, D; Guerreiro, M R; Kremer, E J; Coroadinha, A S; Alves, P M

    2015-01-01

    Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction. PMID:25338917

  3. The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species.

    PubMed

    Dicks, Matthew D J; Guzman, Efrain; Spencer, Alexandra J; Gilbert, Sarah C; Charleston, Bryan; Hill, Adrian V S; Cottingham, Matthew G

    2015-02-25

    Adenovirus vaccine vectors generated from new viral serotypes are routinely screened in pre-clinical laboratory animal models to identify the most immunogenic and efficacious candidates for further evaluation in clinical human and veterinary settings. Here, we show that studies in a laboratory species do not necessarily predict the hierarchy of vector performance in other mammals. In mice, after intramuscular immunization, HAdV-5 (Human adenovirus C) based vectors elicited cellular and humoral adaptive responses of higher magnitudes compared to the chimpanzee adenovirus vectors ChAdOx1 and AdC68 from species Human adenovirus E. After HAdV-5 vaccination, transgene specific IFN-γ(+) CD8(+) T cell responses reached peak magnitude later than after ChAdOx1 and AdC68 vaccination, and exhibited a slower contraction to a memory phenotype. In cattle, cellular and humoral immune responses were at least equivalent, if not higher, in magnitude after ChAdOx1 vaccination compared to HAdV-5. Though we have not tested protective efficacy in a disease model, these findings have important implications for the selection of candidate vectors for further evaluation. We propose that vaccines based on ChAdOx1 or other Human adenovirus E serotypes could be at least as immunogenic as current licensed bovine vaccines based on HAdV-5. PMID:25629523

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that an adenovirus-based FMDV serotype A24 subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1Campos (...

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

    PubMed Central

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

    2015-01-01

    Background Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. Methods HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. Results All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. Conclusions Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted. PMID:26587311

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

    PubMed

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

    2008-03-01

    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients. PMID:18205221

  7. Implications of the innate immune response to adenovirus and adenoviral vectors

    PubMed Central

    Gregory, Seth M; Nazir, Shoab A; Metcalf, Jordan P

    2011-01-01

    Adenovirus (AdV) is a common cause of respiratory illness in both children and adults. Respiratory symptoms can range from those of the common cold to severe pneumonia. Infection can also cause significant disease in the immunocompromised and among immunocompetent subjects in close quarters. Fortunately, infection with AdV in the normal host is generally mild. This is one reason why its initial use as a gene-therapy vector appeared to be so promising. Unfortunately, both innate and adaptive responses to the virus have limited the development of AdV vectors as a tool of gene therapy by increasing toxicity and limiting duration of transgene expression. This article will focus on the innate immune response to infection with wild-type AdV and exposure to AdV gene-therapy vectors. As much of the known information relates to the pulmonary inflammatory response, this organ system will be emphasized. This article will also discuss how that understanding has led to the creation of new vectors for use in gene therapy. PMID:21738557

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

    PubMed Central

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

    2010-01-01

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

  9. Protection of chickens against avian influenza with nonreplicating adenovirus-vectored vaccine.

    PubMed

    Toro, H; Tang, D C

    2009-04-01

    Protective immunity against avian influenza (AI) virus has been elicited in chickens by single-dose in ovo or i.m. vaccination with a replication-competent adenovirus (Ad)-free human Ad vector encoding the AI virus A/Turkey/Wisconsin/68 H5 (AdTW68. H5) or the A/Chicken/New York/94 H7 (AdChNY94. H7) hemagglutinin (HA). The AdTW68.H5-vaccinated chickens were protected against both H5N1 and H5N2 highly pathogenic AI virus challenges. The AdChNY94. H7-vaccinated chickens were protected against an H7N3 highly pathogenic avian influenza virus challenge. Chickens vaccinated in ovo with AdTW68.H5 followed by posthatch i.m. vaccination with AdChNY94.H7 responded to both vaccinations, with robust antibody titers against both the H5 and H7 AI proteins. The use of a synthetic AI H5 HA gene codon optimized to match the tRNA pool found in chicken cells is more potent than the cognate H5 HA gene. Mass administration of this AI vaccine can be streamlined with available robotic in ovo injectors. In addition, Ad5-vectored vaccines can be produced rapidly and the safety margin of the nonreplicating vector is superior to that of a replicating counterpart. Furthermore, this mode of vaccination will not interfere with epidemiological surveys of natural AI infections. Finally, the demonstration that Ad-vectored vaccines can be administered repeatedly without appreciably losing potency highlights the commercial potential of this new class of vaccine in poultry. PMID:19276437

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

    PubMed Central

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

    2015-01-01

    Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies. PMID:26610547

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

    PubMed

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

    2015-11-01

    Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies. PMID:26610547

  12. Characterization of an Oncolytic Adenovirus Vector Constructed to Target the cMet Receptor

    PubMed Central

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

    2015-01-01

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

  13. Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults.

    PubMed

    Green, Christopher A; Scarselli, Elisa; Sande, Charles J; Thompson, Amber J; de Lara, Catherine M; Taylor, Kathryn S; Haworth, Kathryn; Del Sorbo, Mariarosaria; Angus, Brian; Siani, Loredana; Di Marco, Stefania; Traboni, Cinzia; Folgori, Antonella; Colloca, Stefano; Capone, Stefania; Vitelli, Alessandra; Cortese, Riccardo; Klenerman, Paul; Nicosia, Alfredo; Pollard, Andrew J

    2015-08-12

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. PMID:26268313

  14. Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: Toward realistic long-term neurological gene therapy for chronic diseases

    PubMed Central

    Thomas, Clare E.; Schiedner, Gudrun; Kochanek, Stefan; Castro, Maria G.; Löwenstein, Pedro R.

    2000-01-01

    Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8+ cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders. PMID:10840055

  15. Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

    PubMed Central

    Venkatesh, L K; Arens, M Q; Subramanian, T; Chinnadurai, G

    1990-01-01

    The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis. Images PMID:2247444

  16. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer.

    PubMed

    Pickles, R J; McCarty, D; Matsui, H; Hart, P J; Randell, S H; Boucher, R C

    1998-07-01

    Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium. PMID:9621064

  17. Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer

    PubMed Central

    Pickles, Raymond J.; McCarty, Douglas; Matsui, Hirotoshi; Hart, Pádraig J.; Randell, Scott H.; Boucher, Richard C.

    1998-01-01

    Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of αvβ3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium. PMID:9621064

  18. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors.

    PubMed

    Boviatsis, E J; Chase, M; Wei, M X; Tamiya, T; Hurford, R K; Kowall, N W; Tepper, R I; Breakefield, X O; Chiocca, E A

    1994-02-01

    Three vectors derived from retrovirus, herpes simplex virus type 1 (HSV), and adenovirus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a foreign gene (lacZ from Escherichia coli) into cells was more efficient with either the replication-defective retrovirus vector or the replication-conditional thymidine kinase (TK)-deficient HSV vector than with the replication-defective adenovirus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed three main histopathologic findings: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred into several types of endogenous neural cells, as well as into cells within the tumor; (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector, whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors. PMID:8186298

  19. Comparative Analysis of Simian Immunodeficiency Virus Gag-Specific Effector and Memory CD8+ T Cells Induced by Different Adenovirus Vectors

    PubMed Central

    Tan, Wendy G.; Jin, Hyun-Tak; West, Erin E.; Penaloza-MacMaster, Pablo; Wieland, Andreas; Zilliox, Michael J.; McElrath, M. Juliana

    2013-01-01

    Adenovirus (Ad) vectors are widely used as experimental vaccines against several infectious diseases, but the magnitude, phenotype, and functionality of CD8+ T cell responses induced by different adenovirus serotypes have not been compared. To address this question, we have analyzed simian immunodeficiency virus Gag-specific CD8+ T cell responses in mice following vaccination with Ad5, Ad26, and Ad35. Our results show that although Ad5 is more immunogenic than Ad26 and Ad35, the phenotype, function, and recall potential of memory CD8+ T cells elicited by these vectors are substantially different. Ad26 and Ad35 vectors generated CD8+ T cells that display the phenotype and function of long-lived memory T cells, whereas Ad5 vector-elicited CD8+ T cells are of a more terminally differentiated phenotype. In addition, hepatic memory CD8+ T cells elicited by Ad26 and Ad35 mounted more robust recall proliferation following secondary challenge than those induced by Ad5. Furthermore, the boosting potential was higher following priming with alternative-serotype Ad vectors than with Ad5 vectors in heterologous prime-boost regimens. Anamnestic CD8+ T cell responses were further enhanced when the duration between priming and boosting was extended from 30 to 60 days. Our results demonstrate that heterologous prime-boost vaccine regimens with alternative-serotype Ad vectors elicited more functional memory CD8+ T cells than any of the regimens containing Ad5. In summary, these results suggest that alternative-serotype Ad vectors will prove useful as candidates for vaccine development against human immunodeficiency virus type 1 and other pathogens and also emphasize the importance of a longer rest period between prime and boost for generating optimal CD8+ T cell immunity. PMID:23175355

  20. Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

    PubMed Central

    Hehir, K M; Armentano, D; Cardoza, L M; Choquette, T L; Berthelette, P B; White, G A; Couture, L A; Everton, M B; Keegan, J; Martin, J M; Pratt, D A; Smith, M P; Smith, A E; Wadsworth, S C

    1996-01-01

    Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Generation and screening of a large collection of novel simian Adenovirus allows the identification of vaccine vectors inducing potent cellular immunity in humans

    PubMed Central

    Colloca, Stefano; Folgori, Antonella; Ammendola, Virginia; Capone, Stefania; Cirillo, Agostino; Siani, Loredana; Naddeo, Mariarosaria; Grazioli, Fabiana; Esposito, Maria Luisa; Ambrosio, Maria; Sparacino, Angela; Bartiromo, Marta; Meola, Annalisa; Smith, Kira; Kurioka, Ayako; O’Hara, Geraldine A.; Ewer, Katie J.; Hill, Adrian V. S.; Traboni, Cinzia; Barnes, Eleanor; Klenerman, Paul; Cortese, Riccardo; Nicosia, Alfredo

    2013-01-01

    Replication defective Adenovirus vectors based on the human serotype 5 (Ad5) have been shown to induce protective immune responses against diverse pathogens and cancer in animal models and to elicit robust and sustained cellular immunity in humans. However, most humans have anti-Ad5 neutralising antibodies that can impair the immunological potency of such vaccines. Here we show that most other human Adenoviruses from rare serotypes are far less potent as vaccine vectors than Ad5 in mice and non-human primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans we isolated and sequenced over a thousand Adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from different ChAd serotypes and were screened for neutralization by human sera and for ability to grow in human cell lines already approved for clinical studies. Most importantly, we devised a screening strategy to rank the ChAd vectors by immunological potency in mice which predicts their immunogenicity in non-human primates and humans. The vectors studied varied by up to a thousand-fold in potency for CD8 T cell induction in mice. Two of the most potent ChAd vectors were selected for clinical studies as carriers for Malaria and Hepatitis C virus (HCV) genetic vaccines. These ChAd vectors were found to be safe and immunologically potent in Phase I clinical trials thereby validating our screening approach. The ChAd vectors that we have developed represent a large collection of non cross-reactive, potent vectors that can be exploited for diverse vaccine strategies. PMID:22218691

  3. Adenovirus vector induced Innate Immune responses: Impact upon efficacy and toxicity in gene therapy and vaccine applications

    PubMed Central

    Hartman, Zachary C.; Appledorn, Daniel M.; Amalfitano, Andrea

    2013-01-01

    Extensively characterized, modified, and employed for a variety of purposes, Adenovirus (Ad) vectors are generally regarded as having great potential by many applied virologists who wish to manipulate and use viral biology to achieve beneficial clinical outcomes. Despite widespread functional prominence and utility, (i.e.: Ad based clinical trials have begun to progress to critical Phase III levels, it has recently become apparent that investigations regarding the innate immune response to Ads may reveal not only reasons behind previous failures, but also reveal novel insights that will allow for safer, more efficacious uses of this important gene transfer platform. Insights gained by the exploration of Ad induced innate immune responses will likely be most important to the fields of vaccine development, since Ad based vaccines are highly acknowledged as one of the more promising vaccine platforms in development today. Adenovirus is currently known to interact with several different extracellular, intracellular, and membrane bound innate immune sensing systems. Past and recent studies involving manipulation of the Ad infectious cycle as well as use of different mutants have shed light on some of the initiation mechanisms underlying Ad induced immune responses. More recent studies using microarray based analyses, genetically modified cell lines and/or mouse mutants, and advanced generation Ad vectors have revealed important new insights into the scope and mechanism of this cellular defensive response. This review is an attempt to synthesize these studies, update Ad biologists to the current knowledge surrounding these increasingly important issues, as well point areas where future research should be directed. It should also serve as a sobering reality to researchers exploring the use of any gene transfer vector, as to the complexities potentially involved when contemplating use of such vectors for human applications. PMID:18036698

  4. Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

    PubMed

    Sparer, T E; Wynn, S G; Clark, D J; Kaplan, J M; Cardoza, L M; Wadsworth, S C; Smith, A E; Gooding, L R

    1997-03-01

    Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population. PMID:9032363

  5. Adenovirus vaccine vectors expressing hepatitis B surface antigen: importance of regulatory elements in the adenovirus major late intron.

    PubMed

    Mason, B B; Davis, A R; Bhat, B M; Chengalvala, M; Lubeck, M D; Zandle, G; Kostek, B; Cholodofsky, S; Dheer, S; Molnar-Kimber, K

    1990-08-01

    Adenovirus types 4 and 7 are currently used as live oral vaccines for prevention of acute respiratory disease caused by these adenovirus serotypes. To investigate the concept of producing live recombinant vaccines using these serotypes, adenovirus types 4 (Ad4) and 7 (Ad7) were constructed that produce HBsAg upon infection of cell cultures. Ad4 recombinants were constructed that express HBsAg from a cassette inserted 135 bp from the right-hand terminus of the viral genome. The cassette contained the Ad4 major late promoter followed by leader 1 of the tripartite leader, the first intervening sequence between leaders 1 and 2, leaders 2 and 3, the HBsAg gene, and tandem polyadenylation signals from the Ad4 E3B and hexon genes. Using this same cassette, a series of Ad4 recombinants expressing HBsAg were constructed with deletions in the intervening sequence between leaders 1 and 2 to evaluate the contribution of the downstream control elements more precisely. Inclusion of regions located between +82 and +148 as well as +148 and +232 resulted in increases in expression levels of HBsAg in A549-infected cells by 22-fold and 44-fold, respectively, over the levels attained by an adenovirus recombinant retaining only sequences from +1 to +82, showing the importance of these elements in the activation of the major late promoter during the course of a natural Ad4 viral infection. Parallel increases were also observed in steady-state levels of cytoplasmic HBsAg-specific mRNA. When similar Ad7 recombinant viruses were constructed, these viruses also expressed 20-fold more HBsAg due to the presence of the intron. All Ad4 and Ad7 recombinants produced HBsAg particles containing gp27 and p24 which were secreted in the medium. When dogs were immunized intratracheally with one of these Ad7 recombinants, they seroconverted to both Ad7 and HBsAg to a high level. PMID:2371766

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

    SciTech Connect

    Wang Danher; Hevey, Michael; Juompan, Laure Y.; Trubey, Charles M.; Raja, Nicholas U.; Deitz, Stephen B.; Woraratanadharm, Jan; Luo Min; Yu Hong; Swain, Benjamin M.; Moore, Kevin M.; Dong, John Y. . E-mail: dongj@genphar.com

    2006-09-30

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

    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

  9. Methods and clinical development of adenovirus-vectored vaccines against mucosal pathogens

    PubMed Central

    Afkhami, Sam; Yao, Yushi; Xing, Zhou

    2016-01-01

    Adenoviruses represent the most widely used viral-vectored platform for vaccine design, showing a great potential in the fight against intracellular infectious diseases to which either there is a lack of effective vaccines or the traditional vaccination strategy is suboptimal. The extensive understanding of the molecular biology of adenoviruses has made the new technologies and reagents available to efficient generation of adenoviral-vectored vaccines for both preclinical and clinical evaluation. The novel adenoviral vectors including nonhuman adenoviral vectors have emerged to be the further improved vectors for vaccine design. In this review, we discuss the latest adenoviral technologies and their utilization in vaccine development. We particularly focus on the application of adenoviral-vectored vaccines in mucosal immunization strategies against mucosal pathogens including Mycobacterium tuberculosis, flu virus, and human immunodeficiency virus. PMID:27162933

  10. Development of Dual-Activity Vectors by Co-Envelopment of Adenovirus and SiRNA in Artificial Lipid Bilayers

    PubMed Central

    Yilmazer, Açelya; Tian, Bowen; Kostarelos, Kostas

    2014-01-01

    Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. PMID:25501573

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

    PubMed

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

    2016-08-01

    P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. PMID:27286705

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

    PubMed

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

    2014-04-01

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

  13. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector.

    PubMed

    Toro, Haroldo; Tang, De-chu C; Suarez, David L; Sylte, Matt J; Pfeiffer, Jennifer; Van Kampen, Kent R

    2007-04-12

    Protective immunity against avian influenza virus was elicited in chickens by single-dose in ovo vaccination with a non-replicating human adenovirus vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 (89% hemagglutinin homology; 68% protection) and H5N2 (94% hemagglutinin homology; 100% protection) highly pathogenic avian influenza virus challenges. This vaccine can be mass-administered using available robotic in ovo injectors which provide a major advantage over current vaccination regimens. In addition, this class of adenovirus-vectored vaccines can be produced rapidly with improved safety since they do not contain any replication-competent adenoviruses. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural avian influenza virus infections. PMID:17055126

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Cycles of Transient High-Dose Cyclophosphamide Administration and Oncolytic Adenovirus Vector Intratumoral Injection for Long Term Tumor Suppression in Syrian Hamsters

    PubMed Central

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

    2014-01-01

    Immune responses against oncolytic adenovirus (Ad) vectors are thought to limit vector anti-tumor efficacy. In Syrian hamsters, which are immunocompetent and whose tumors and normal tissues are permissive for replication of Ad5-based oncolytic Ad vectors, treating with high-dose cyclophosphamide to suppress the immune system and exert chemotherapeutic effects enhances Ad vector anti-tumor efficacy. However, long term cyclophosphamide treatment and immunosuppression can lead to anemia and vector spread to normal tissues. Here we employed three cycles of transient high-dose cyclophosphamide administration plus intratumoral injection of the oncolytic Ad vector VRX-007 followed by withdrawal from cyclophosphamide. Each cycle lasted 4-6 weeks. This protocol allowed the hamsters to remain healthy so the study could be continued for ~100 days. The tumors were very well suppressed throughout the study. With immunocompetent hamsters, the vector retarded tumor growth initially, but after 3-4 weeks the tumors resumed rapid growth and further injections of vector were ineffective. Preimmunization of the hamsters with Ad5 prevented vector spillover from the tumor to the liver yet still allowed for effective long term anti-tumor efficacy. Our results suggest that a clinical protocol might be developed with cycles of transient chemotherapy plus intratumoral vector injection to achieve significant anti-tumor efficacy while minimizing the side effects of cytostatic treatment. PMID:24722357

  17. Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity.

    PubMed

    Gabitzsch, Elizabeth S; Xu, Younong; Yoshida, Lois H; Balint, Joseph; Amalfitano, Andrea; Jones, Frank R

    2009-10-30

    Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naïve and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation. PMID:19559110

  18. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

    PubMed Central

    Askanas, V; McFerrin, J; Baqué, S; Alvarez, R B; Sarkozi, E; Engel, W K

    1996-01-01

    As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8577761

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

    SciTech Connect

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

    2006-06-05

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

  20. Adenovirus serotype 5 vectored foot-and-mouth disease subunit vaccines: the first decade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we present the results of the first decade of development of a replication-defective human adenovirus (Ad5) containing the capsid and 3C protease coding regions of foot-and-mouth disease virus (FMDV) as a vaccine candidate. In proof-of concept studies we demonstrated that a single inoculation w...

  1. Construction of recombinant adenovirus Ad-rat PLCg2-shRNA and successful suppression of PLCg2 expression in BRL-3A cells.

    PubMed

    Chen, X G; Lv, Q X; Zhou, X Q

    2016-01-01

    Phospholipase Cg2 (PLCg2) induces apoptosis of immune and tumor cells; however, it remains unclear whether PLCg2 promotes hepatocyte apoptosis during liver regeneration (LR). Therefore, to establish a framework for further exploring the function of PLCg2, we generated recombinant adenoviruses carrying a template encoding short hairpin (sh)-RNA targeting PLCg2 (Ad-PLCg2-shRNA), which were used to silence the expression of PLCg2 in BRL-3A cells. First, three pairs of PLCg2-shRNAs were designed, synthesized, and cloned into a shuttle vector, pHBAd-U6-GFP, after annealing. The recombinant shuttle plasmids were co-transfected with the backbone vector pHBAd-BHG into HK293 cells to package the recombinant Ad-PLCg2-shRNAs used to infect BRL-3A cells. Infection efficiency was monitored by observing the number of GFP-positive cells under a fluorescent microscope. To determine the recombinant adenoviruses with the highest silencing efficiency, levels of PLCg2 mRNA were evaluated by qRT-PCR. DNA sequencing confirmed that the correct shRNA coding sequences were inserted into the shuttle vectors and adenoviral plasmids. The titers of three recombinant adenoviruses were at least 1 x 10(10) PFU/mL. The most effective adenoviral construct, with interference efficiency of 77%, was determined by qRT-PCR. These results show that a recombinant adenovirus, Ad-PLCg2-shRNA, was developed and was effective at silencing the rat PLCg2 gene. This construct may contribute to the study of PLCg2 in hepatocyte apoptosis during LR. PMID:27323081

  2. Partial protection against H5N1 influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein.

    PubMed

    Roy, Soumitra; Kobinger, Gary P; Lin, Jianping; Figueredo, Joanita; Calcedo, Roberto; Kobasa, Darwyn; Wilson, James M

    2007-09-28

    The development of adenoviral vectors based on non-human serotypes such as the chimpanzee adenovirus simian adenovirus 24 (AdC7) may allow for their utilization in populations harboring neutralizing antibodies to common human serotypes. Because adenoviral vectors can be used to generate potent T cell responses, they may be useful as vaccines against pandemic influenza such as may be caused by the H5N1 strains that are currently endemic in avian populations. The influenza nucleoprotein (NP) is known to provide MHC Class I restricted epitopes that are effective in evoking a cytolytic response. Because there is only low sequence variation in NP sequences between different influenza strains, a T cell vaccine may provide heterosubtypic protection against a spectrum of influenza A strains. An AdC7 vector expressing the influenza A/Puerto Rico/8/34 NP was tested for its efficacy in protecting BALB/c mice against two H5N1 strains and compared to a conventional human adenovirus serotype 5 vaccine. The AdC7 NP vaccine elicited a strong anti-NP T cell response. When tested in a mouse challenge model, there was improved survival following challenge with two strains of H5N1 that have caused human outbreaks, Vietnam/1203/04 and Hong Kong/483/97, although the improved survival reached statistical significance only with the strain from Vietnam. PMID:17728024

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

    PubMed Central

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

    1996-01-01

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

  4. Type I interferon-dependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression.

    PubMed

    Johnson, Matthew J; Björkström, Niklas K; Petrovas, Constantinos; Liang, Frank; Gall, Jason G D; Loré, Karin; Koup, Richard A

    2014-02-01

    Vaccines constructed from rare-serotype recombinant adenovirus vectors (rAd) such as rAd serotype 28 (rAd28) and rAd35 are currently being explored as alternatives to rAd5-based vaccines because they circumvent the problems with pre-existing immunity that complicate the effectiveness of rAd5 vaccines. However, previous work has demonstrated that the immunogenicity of rAd28 and rAd35 is substantially lower than rAd5. Here we show that rAd28 and rAd35 increase apoptosis of antigen presenting cells (APCs), such as monocytes, relative to rAd5 and mock infected controls. APCs undergoing apoptosis showed an increased loss of vector-insert expression. Loss of vector-insert expression correlated with activation of NK cells, which resulted in apoptosis of co-cultured monocytes. Finally, we show that activation of NK cells is dependent on IFNα which is produced by exposure to rAd28 or rAd35, but not to rAd5. Taken together, these data demonstrate that IFNα-induced activation of NK cells leads to increased monocyte apoptosis and subsequent vector-insert loss. This may be a possible mechanism that results in reduced immunogenicity of rAd28 and rAd35-based vectors. PMID:24325826

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

    PubMed Central

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

    2013-01-01

    Background Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. Methodology/Principal Findings As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. Conclusions/Significance These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers. PMID:23383334

  6. Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors.

    PubMed

    Okada, Yuka; Okada, Naoki; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki; Kanehira, Makiko; Nishino, Naoko; Takahashi, Koichi; Mizuno, Nobuyasu; Hayakawa, Takao; Mayumi, Tadanori

    2002-03-01

    Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma. PMID:11809531

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

    The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines. PMID:22432020

  9. Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles

    PubMed Central

    Walkiewicz, Marcin P.; Morral, Nuria; Engel, Daniel A.

    2009-01-01

    Summary Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72 kDa DNA binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a one-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay. PMID:19406166

  10. Infection by retroviral vectors outside of their host range in the presence of replication-defective adenovirus.

    PubMed Central

    Adams, R M; Wang, M; Steffen, D; Ledley, F D

    1995-01-01

    Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease. PMID:7853530

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

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

    PubMed

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

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells. PMID:26280081

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

    PubMed Central

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

    2015-01-01

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

  14. An investigation of adverse effects caused by the injection of high-dose TNFalpha-expressing adenovirus vector into established murine melanoma.

    PubMed

    Okada, Y; Okada, N; Mizuguchi, H; Hayakawa, T; Mayumi, T; Mizuno, N

    2003-04-01

    We previously reported that RGD fiber-mutant adenovirus vector carrying human TNFalpha cDNA (AdRGD-TNFalpha) could more effectively induce mouse B16 BL6 melanoma regression than conventional Ad-TNFalpha on intratumoral injection at less than 10(9) vector particles (VP). Although mice treated with either Ad type at 10(10) VP showed remarkable tumor regression due to hemolytic necrosis, severe adverse effects including extreme reduction in body weight were also induced by Ad treatment. Here, we attempted to elucidate the cause of the adverse effects to optimize the application of AdRGD-TNFalpha. More than 99% of systemically administered Ad accumulated in the liver, and the rate of Ad leakage into systemic circulation from the B16 BL6 tumors injected with AdRGD or conventional Ad at 10(10) VP was about 1% of the administered VP. Although the leaked Ad did not directly induce hepatotoxicity or body weight reduction, excessive TNFalpha produced in the tumors leaked into the blood at high concentrations and caused systemic inflammation, tissue denaturation, and body weight reduction in mice injected intratumorally with AdRGD-TNFalpha or Ad-TNFalpha at 10(10) VP. Our results demonstrated that an exact AdRGD-TNFalpha dosage must be determined to prevent TNFalpha leakage from tumors into systemic circulation, thereby enabling safe application of AdRGD-TNFalpha to clinical melanoma gene therapy in the future. PMID:12692598

  15. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

    PubMed

    Ehrke-Schulz, Eric; Zhang, Wenli; Schiwon, Maren; Bergmann, Thorsten; Solanki, Manish; Liu, Jing; Boehme, Philip; Leitner, Theo; Ehrhardt, Anja

    2016-01-01

    High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented. PMID:26863087

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

    PubMed Central

    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.

    2005-01-01

    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-γ–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 β-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. PMID:15841217

  17. Heterologous prime-boost immunization regimens using adenovirus vector and virus-like particles induce broadly neutralizing antibodies against H5N1 avian influenza viruses.

    PubMed

    Lin, Shih-Chang; Liu, Wen-Chun; Lin, Yu-Fen; Huang, Yu-Hsuan; Liu, Jin-Hwang; Wu, Suh-Chin

    2013-11-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to trigger severe diseases in poultry and humans, prompting efforts to develop an effective vaccine. Toward that goal, we constructed a recombinant adenovirus vector encoding influenza hemagglutin (rAd-HA) and a flagellin-containing virus-like particle (FliC-VLP). Using a murine model, we investigated a heterologous prime-boost vaccination regimen combining these two vectors. Our results indicate that priming with the rAd-HA vector followed by a FliC-VLP booster induced the highest HA-specific total IgG, IgG1and IgG2a. Maximum neutralizing antibody titers against homologous and heterologous clades of H5N1 virus strains and hemagglutination inhibition resulted from the heterologous vaccination strategy. Our results are likely to contribute to the development of more effective H5N1 vaccines. PMID:23813782

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

    PubMed

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

    2014-11-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FMDV challenge by 7 days post-vaccination. However, since relatively large amounts of Ad5-CI-A24-2B are required to induce protection this strategy could be costly for livestock production. Poly ICLC is a synthetic double stranded RNA that activates multiple innate and adaptive immune pathways. In this study, we have tested for the first time, the adjuvant effect of poly ICLC in combination with Ad5-CI-A24-2B in swine. We found that the combination resulted in a reduction of the vaccine protective dose by 80-fold. Interestingly, the lowest dose of Ad5-CI-A24-2B plus 1mg of poly ICLC protected animals against challenge even in the absence of detectable FMDV-specific neutralizing antibodies at the time of challenge. PMID:25216089

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

    PubMed Central

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

    2016-01-01

    Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-13C]glucose and [U-13C]glutamine, we apply for the first time 13C-Metabolic flux analysis (13C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and 13C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. 13C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production. PMID:27004747

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

    PubMed

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

    2013-01-01

    The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy. PMID:23531212

  1. Adenovirus-vectored drug-vaccine duo as a rapid-response tool for conferring seamless protection against influenza.

    PubMed

    Zhang, Jianfeng; Tarbet, E Bart; Feng, Tsungwei; Shi, Zhongkai; Van Kampen, Kent R; Tang, De-chu C

    2011-01-01

    Few other diseases exert such a huge toll of suffering as influenza. We report here that intranasal (i.n.) administration of E1/E3-defective (ΔE1E3) adenovirus serotype 5 (Ad5) particles rapidly induced an anti-influenza state as a means of prophylactic therapy which persisted for several weeks in mice. By encoding an influenza virus (IFV) hemagglutinin (HA) HA1 domain, an Ad5-HA1 vector conferred rapid protection as a prophylactic drug followed by elicitation of sustained protective immunity as a vaccine for inducing seamless protection against influenza as a drug-vaccine duo (DVD) in a single package. Since Ad5 particles induce a complex web of host responses, which could arrest influenza by activating a specific arm of innate immunity to impede IFV growth in the airway, it is conceivable that this multi-pronged influenza DVD may escape the fate of drug resistance that impairs the current influenza drugs. PMID:21818346

  2. Immortalisation of human oesophageal epithelial cells by a recombinant SV40 adenovirus vector.

    PubMed Central

    Inokuchi, S.; Handa, H.; Imai, T.; Makuuchi, H.; Kidokoro, M.; Tohya, H.; Aizawa, S.; Shimamura, K.; Ueyama, Y.; Mitomi, T.

    1995-01-01

    We introduced the origin-defective SV40 early gene into cultured human oesophageal epithelial cells by infection of a recombinant SV40 adenovirus vector. The virus-infected cells formed colonies 3-4 weeks after infection in medium containing fetal calf serum. When the cells derived from 'serum-resistant' colonies were then maintained in the serum-free medium with a low calcium ion concentration, some of them passed the cell crisis and kept growing for over 12 months. These cells, regarded as immortalised cells, resembled the primarily cultured oesophageal epithelial cells in morphology and had some of their original characteristics. Treatment of the cells with a high calcium concentration induced phenotypic changes. These cells still responded to transforming growth factor beta. When the immortalised cells were injected into severe combined immunodeficient mice, they transiently formed epithelial cysts, although the typical differentiation pattern of the oesophageal epithelium was not observed. These cysts regressed within 2 months without development into tumours. The results indicated that human oesophageal epithelial cells were reproducibly immortalised by infection with a recombinant SV40 adenovirus vector at relatively high efficiency. The immortalised cells should be useful in studies on oesophageal carcinogenesis and in assessing the cooperative effects with other oncogene products or carcinogens. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7536023

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

    PubMed Central

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

    2015-01-01

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

  4. Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

    PubMed Central

    Teramoto, S.; Bartlett, J. S.; McCarty, D.; Xiao, X.; Samulski, R. J.; Boucher, R. C.

    1998-01-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  5. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

    PubMed

    Teramoto, S; Bartlett, J S; McCarty, D; Xiao, X; Samulski, R J; Boucher, R C

    1998-11-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  6. Comparative immunogenicity of recombinant adenovirus-vectored vaccines expressing different forms of hemagglutinin (HA) proteins from the H5 serotype of influenza A viruses in mice.

    PubMed

    Hu, Xiangjing; Meng, Weixu; Dong, Zhenyuan; Pan, Weiqi; Sun, Caijun; Chen, Ling

    2011-01-01

    Recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 viruses in poultry and their subsequent transmission to humans have highlighted an urgent need to develop preventive vaccines in the event of a pandemic. In this paper we constructed recombinant adenovirus (rAd)-vectored influenza vaccines expressing different forms of H5 hemagglutinin (HA) from the A/Vietnam/1194/04 (VN/1194/04) virus, a wild-type HA, a sequence codon-optimized HA and a transmembrane (TM) domain-truncated HA. Compared to the rAd vectors expressing the wild-type HA (rAd-04wtHA) and the TM-truncated form of HA (rAd-04optHA-dTM), the rAd vectored vaccine with the sequence codon-optimized HA (rAd-04optHA) showed a tendency to induce much higher hemagglutinin inhibition (HI) antibody titers in mice immunized with a prime-boost vaccine. Furthermore, administration of the rAd-04optHA vaccine to mice could elicit cross-reactive immune responses against the antigenically distinct HK/482/97 virus. Additionally, we constructed another vector containing the codon-optimized HA of the A/Hong Kong/482/97 (HK/482/97) virus. Administration of a bivalent immunization formulation including the rAd-04optHA and rAd-97optHA vaccines to mice induced a stronger immune response against HK/482/97 virus than the monovalent formulation. Taken together, these findings may have some implications for the development of rAd-vectored vaccines in the event of the pandemic spread of HPAI. PMID:20883733

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  8. Vaccines within vaccines: the use of adenovirus types 4 and 7 as influenza vaccine vectors.

    PubMed

    Weaver, Eric A

    2014-01-01

    Adenovirus Types 4 and 7 (Ad4 and Ad7) are associated with acute respiratory distress (ARD). In order to prevent widespread Ad-associated ARD (Ad-ARD) the United States military immunizes new recruits using a safe and effective lyophilized wildtype Ad4 and Ad7 delivered orally in an enteric-coated capsule. We cloned Ad4 and Ad7 and modified them to express either a GFP-Luciferase (GFPLuc) fusion gene or a centralized influenza H1 hemagglutinin (HA1-con). BALB/c mice were injected with GFPLuc expressing viruses intramuscularly (i.m.) and intranasally (i.n.). Ad4 induced significantly higher luciferase expression levels as compared with Ad7 by both routes. Ad7 transduction was restored using a human CD46+ transgenic mouse model. Mice immunized with serial dilutions of viruses expressing the HA1-con influenza vaccine gene were challenged with 100 MLD 50 of influenza virus. Ad4 protected BALB/c mice at a lower dose by i.m. immunization as compared with Ad7. Unexpectedly, there was no difference in protection by i.n. immunization. Although Ad7 i.m. transduction was restored in CD46+ transgenic mice, protection against influenza challenge required even higher doses as compared with the BALB/c mice. However, Ad7 i.n. immunized CD46+ transgenic mice were better protected as compared with Ad4. Interestingly, the restoration of Ad7 transduction in CD46+ mice did not increase vaccine efficacy and indicates that Ad7 may transduce a different subset of cells through alternative receptors in the absence of CD46. These data indicate that both Ad4 and Ad7 can effectively induce anti-H1N1 immunity against a heterologous challenge using a centralized H1 gene. Future studies in non-human primates or human clinical trials will determine the overall effectiveness of Ad4 and Ad7 as vaccines for influenza. PMID:24280656

  9. Virucidal effects of rodent cage-cleaning practices on the viability of adenovirus vectors.

    PubMed

    Porter, Jacqueline D; Lyons, Russette M

    2002-09-01

    Human adenoviruses and adenoviral vectors are classified as Risk Group 2 agents and require BSL2 containment and practices. An additional consideration in using adenoviruses and viral vectors in laboratory animal studies is the possible transmission of these agents to other animals and/or personnel as a result of viral shedding in animal urine and feces. When handling BSL2 agents, cage-wash staff are required to wear appropriate personnel protective equipment, including scrubs, Tyvek suit, hair covering, dust mask, shoes covers, and gloves. Current decontamination procedures are to bag and autoclave soiled rodent cages containing bedding prior to washing in the cage washer to prevent possible adenoviral transmission. However, the practice of autoclaving softens the polycarbonate-based rodent cages, allowing damaging agents or conditions to affect the integrity of the plastic and degrade the cages. The objective of this study was to determine whether current rodent cage-cleaning practices produced virucidal effects for use in lieu of or prior to autoclaving the cages. We found that heating an Av3GFP vector in a test tube to a temperature of 74 degrees C (165 degrees F) for 6 min conditions equivalent to those of the cage washer resulted in greater than an 11-log reduction in infectivity of the vector as evaluated by its cytopathic effect on cells. The combination of heating and a liquid, phosphate-free alkaline detergent produced the same reduction in vector infectivity. However, common cage-cleaning solutions alone possessed no virucidal activity. The high temperatures used in cage-washing procedures alone or in combination with a cleaning solution reduced or eliminated the risk of transmission from viral shedding through urine and feces even at vector concentrations far greater than would ever be expected to be present. Autoclaving cages diminishes the stability and integrity of the polycarbonate cages without providing a further reduction in the risk of virus or

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

    PubMed Central

    CHEN, XUEMEI; LIU, DI; WANG, JUNFU; SU, QINGHONG; ZHOU, PENG; LIU, JINSHENG; LUAN, MENG; XU, XIAOQUN

    2014-01-01

    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. PMID:24527085

  11. Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob

    PubMed Central

    Krasnykh, Victor; Dmitriev, Igor; Mikheeva, Galina; Miller, C. Ryan; Belousova, Natalya; Curiel, David T.

    1998-01-01

    The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies. PMID:9499035

  12. Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob.

    PubMed

    Krasnykh, V; Dmitriev, I; Mikheeva, G; Miller, C R; Belousova, N; Curiel, D T

    1998-03-01

    The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies. PMID:9499035

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

    PubMed

    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 < 0.01) than with either HAd-H5HA or 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. PMID:18301400

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

    PubMed

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

    2015-06-22

    There is an urgent need for improvements in vaccine delivery technologies. This is particularly pertinent for vaccination programmes within regions of limited resources, such as those required for adequate provision for disposal of used needles. Microneedles are micron-sized structures that penetrate the stratum corneum of the skin, creating temporary conduits for the needle-free delivery of drugs or vaccines. Here, we aimed to investigate immunity induced by the recombinant simian adenovirus-vectored vaccine ChAd63.ME-TRAP; currently undergoing clinical assessment as a candidate malaria vaccine, when delivered percutaneously by silicon microneedle arrays. In mice, we demonstrate that microneedle-mediated delivery of ChAd63.ME-TRAP induced similar numbers of transgene-specific CD8(+) T cells compared to intradermal (ID) administration with needle-and-syringe, following a single immunisation and after a ChAd63/MVA heterologous prime-boost schedule. When mice immunised with ChAd63/MVA were challenged with live Plasmodium berghei sporozoites, microneedle-mediated ChAd63.ME-TRAP priming demonstrated equivalent protective efficacy as did ID immunisation. Furthermore, responses following ChAd63/MVA immunisation correlated with a specific design parameter of the array used ('total array volume'). The level of transgene expression at the immunisation site and skin-draining lymph node (dLN) was also linked to total array volume. These findings have implications for defining silicon microneedle array design for use with live, vectored vaccines. PMID:25839104

  15. Generation of Neutralizing Monoclonal Antibodies against a Conformational Epitope of Human Adenovirus Type 7 (HAdv-7) Incorporated in Capsid Encoded in a HAdv-3-Based Vector

    PubMed Central

    Li, Xiao; Zhou, Zhichao; Li, Chenyang; Zhou, Rong

    2014-01-01

    The generation of monoclonal antibodies (MAbs) by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5) of adenovirus type 7 (HAdv-7) was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA) analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses. PMID:25054273

  16. Core labeling of adenovirus with EGFP

    SciTech Connect

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

    2006-08-01

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

  17. Preventing Spontaneous Genetic Rearrangements in the Transgene Cassettes of Adenovirus Vectors

    PubMed Central

    Cottingham, Matthew G.; Carroll, Fionnadh; Morris, Susan J.; Turner, Alison V.; Vaughan, Aisling M.; Kapulu, Melissa C.; Colloca, Stefano; Siani, Loredana; Gilbert, Sarah C.; Hill, Adrian V.S.

    2016-01-01

    First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture. PMID:22252512

  18. Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.

    PubMed

    Krutzke, L; Prill, J M; Engler, T; Schmidt, C Q; Xu, Z; Byrnes, A P; Simmet, T; Kreppel, F

    2016-08-10

    The biodistribution of adenovirus type 5 (Ad5) vector particles is heavily influenced by interaction of the particles with plasma proteins, including coagulation factor X (FX), which binds specifically to the major Ad5 capsid protein hexon. FX mediates hepatocyte transduction by intravenously-injected Ad5 vectors and shields vector particles from neutralization by natural antibodies and complement. In mice, mutant Ad5 vectors that are ablated for FX-binding become detargeted from hepatocytes, which is desirable for certain applications, but unfortunately such FX-nonbinding vectors also become sensitive to neutralization by mouse plasma proteins. To improve the properties of Ad5 vectors for systemic delivery, we developed a strategy to replace the natural FX shield by a site-specific chemical polyethylene glycol shield. Coupling of polyethylene glycol to a specific site in hexon hypervariable region 1 yielded vector particles that were protected from neutralization by natural antibodies and complement although they were unable to bind FX. These vector particles evaded macrophages in vitro and showed significantly improved pharmacokinetics and hepatocyte transduction in vivo. Thus, site-specific shielding of Ad5 vectors with polyethylene glycol rendered vectors FX-independent and greatly improved their properties for systemic gene therapy. PMID:27302248

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

    PubMed Central

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

    2013-01-01

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

  20. Group D Adenoviruses Infect Primary Central Nervous System Cells More Efficiently than Those from Group C

    PubMed Central

    Chillon, Miguel; Bosch, Assumpció; Zabner, Joseph; Law, Lane; Armentano, Donna; Welsh, Michael J.; Davidson, Beverly L.

    1999-01-01

    Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector. PMID:9971839

  1. Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species

    PubMed Central

    Zahn, Roland; Gillisen, Gert; Roos, Anna; Koning, Marina; van der Helm, Esmeralda; Spek, Dirk; Weijtens, Mo; Grazia Pau, Maria; Radošević, Katarina; Weverling, Gerrit Jan; Custers, Jerome; Vellinga, Jort; Schuitemaker, Hanneke; Goudsmit, Jaap; Rodríguez, Ariane

    2012-01-01

    Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years. PMID:23236343

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

    PubMed

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

    2012-01-01

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

  3. Canine adenovirus based rabies vaccines.

    PubMed

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

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control. PMID:18634509

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

    PubMed Central

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

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models. PMID:24124483

  5. Evaluation of polymer shielding for adenovirus serotype 6 (Ad6) for systemic virotherapy against human prostate cancers

    PubMed Central

    Nguyen, Tien V; Heller, Greg J; Barry, Mary E; Crosby, Catherine M; Turner, Mallory A; Barry, Michael A

    2016-01-01

    Oncolytic viruses hold promise as “self-amplifying” cancer therapies wherein a virally killed cell can produce thousands of new viral “drugs” that can kill more cancer cells. Adenoviruses (Ads) are one family of oncolytic viruses. Most human studies have used human Ad serotype 5 (Ad5). Unfortunately, most patients are already immune to Ad5 increasing the likelihood that the agent will be neutralized if used as a cancer therapy. In this work, lower seroprevalence Ad6 was tested as a systemic therapy for prostate cancer. Ad5 and Ad6 were injected intravenously a single time in nude mice bearing human prostate tumors, and toxicity and efficacy were assessed. Ad6 was chemically shielded with polyethylene glycol (PEG) to test if this would further improve its pharmacology. Ad6 produced 30-fold lower liver damage and less toxicity than Ad5. Ad6 significantly repressed the growth of androgen-resistant human DU145 prostate tumors and androgen-sensitive LNCaP tumors after single intravenous injection. PEGylation did not change virus distribution, but blunted liver damage and cytokine production by Ad6. PEGylated Ad6 eradicated LNCaP tumors and maintained body mass, but lost potency against the more challenging DU145 tumors. These and other data suggest that low seroprevalent Ad6 has better efficacy and safety than the benchmark oncolytic virus Ad5 for systemic therapy of prostate cancer. These data also indicate that PEGylation may improve Ad6 safety, but that this shielding may reduce oncolytic efficacy after intravenous treatment. PMID:26900598

  6. High-level production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors

    PubMed Central

    Hu, Yani; O’Boyle, Kaitlin; Palmer, Donna; Ng, Philip; Sutton, Richard E

    2015-01-01

    Gene transfer vectors based upon human immunodeficiency virus type 1 (HIV) are widely used in bench research applications and increasingly in clinical investigations, both to introduce novel genes but also to reduce expression of unwanted genes of the host and pathogen. At present, the vast majority of HIV-based vector supernatants are produced in 293T cells by cotransfection of up to five DNA plasmids, which is subject to variability and difficult to scale. Here we report the development of a HIV-based vector production system that utilizes helper-dependent adenovirus (HDAd). All necessary HIV vector components were inserted into one or more HDAds, which were then amplified to very high titers of ~1013 vp/ml. These were then used to transduce 293-based cells to produce HIV-based vector supernatants, and resultant VSV G-pseudotyped lentiviral vector (LV) titers and total IU were 10- to 30-fold higher, compared to plasmid transfection. Optimization of HIV-based vector production depended upon maximizing expression of all HIV vector components from HDAd. Supernatants contained trace amounts of HDAd but were free of replication-competent lentivirus. This production method should be applicable to other retroviral vector systems. Scalable production of HIV-based vectors using this two-step procedure should facilitate their clinical advancement. PMID:26029715

  7. Adenovirus 5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part A: Safety and Immunogenicity in Seronegative Adults

    PubMed Central

    Sedegah, Martha; Tamminga, Cindy; McGrath, Shannon; House, Brent; Ganeshan, Harini; Lejano, Jennylynn; Abot, Esteban; Banania, Glenna J.; Sayo, Renato; Farooq, Fouzia; Belmonte, Maria; Manohar, Nalini; Richie, Nancy O.; Wood, Chloe; Long, Carole A.; Regis, David; Williams, Francis T.; Shi, Meng; Chuang, Ilin; Spring, Michele; Epstein, Judith E.; Mendoza-Silveiras, Jose; Limbach, Keith; Patterson, Noelle B.; Bruder, Joseph T.; Doolan, Denise L.; King, C. Richter; Soisson, Lorraine; Diggs, Carter; Carucci, Daniel; Dutta, Sheetij; Hollingdale, Michael R.; Ockenhouse, Christian F.; Richie, Thomas L.

    2011-01-01

    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 malaria vaccine

  8. Preferable sites and orientations of transgene inserted in the adenovirus vector genome: The E3 site may be unfavorable for transgene position

    PubMed Central

    Suzuki, M; Kondo, S; Pei, Z; Maekawa, A; Saito, I; Kanegae, Y

    2015-01-01

    The adenovirus vector (AdV) can carry two transgenes in its genome, the therapeutic gene and a reporter gene, for example. The E3 insertion site has often been used for the expression of the second transgene. A transgene can be inserted at six different sites/orientations: E1, E3 and E4 sites, and right and left orientations. However, the best combination of the insertion sites and orientations as for the titers and the expression levels has not sufficiently been studied. We attempted to construct 18 AdVs producing GFP or LacZ gene driven by the EF1α promoter and Cre gene driven by the α-fetoprotein promoter. The AdV containing GFP gene at E3 in the rightward orientation (GFP-E3R) was not available. The LacZ-E3R AdV showed 20-fold lower titer and 50-fold lower level of fiber mRNA than the control E1L AdV. Notably, we found four aberrantly spliced mRNAs in the LacZ-E3L/R AdVs, probably explaining their very low titers. Although the transgene expression levels in the E4R AdVs were about threefold lower than those in the E1L AdVs, their titers are comparable with that of E1L AdVs. We concluded that E1L and E4R sites/orientations are preferable for expressing the main target gene and a second gene, respectively. PMID:25588742

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

    PubMed Central

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

    2015-01-01

    Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes. PMID:24607662

  10. [Transcatheter delivery of recombinant adenovirus vector containing exogenous aquaporin gene in treatment of Sjögren's syndrome].

    PubMed

    Hong, H E; Jieqiong, Zhang; Yan, Fan; Xiaoshuang, Sun; Yuhao, Zhu

    2016-05-25

    Sjögren's syndrome is a kind of autoimmune disease, whose main clinical symptoms are dry mouth, dry eye and chronic parotid glandular inflammation. The conservative treatments include artificial tears or saliva,oral administration of corticosteroids,and immunosuppressantsl with limited effectiveness. Along with the development of molecular biology, vast attentions are being paid to researches on gene therapy for Sjögren's syndrome, hopefully to bring gospel to patients with Sjögren's syndrome. This article reviews the recent research progresses on transcatheter delivery of recombinant adenovirus vector with aquaporin gene in experimental treatment of Sjögren's syndrome. PMID:27045247

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

    PubMed Central

    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

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

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    Anderson, C.W.

    1981-05-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. A Phase I Clinical Trial of Ad5.SSTR/TK.RGD, a Novel Infectivity-Enhanced Bicistronic Adenovirus, in Patients with Recurrent Gynecologic Cancer

    PubMed Central

    Kim, Kenneth H.; Dmitriev, Igor; O’Malley, Janis P.; Wang, Minghui; Saddekni, Souheil; You, Zhiying; Preuss, Meredith A.; Harris, Raymond D.; Aurigemma, Rosemarie; Siegal, Gene P.; Zinn, Kurt R.; Curiel, David T.; Alvarez, Ronald D.

    2014-01-01

    Purpose Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus expressing a therapeutic thymidine kinase suicide gene and a somatostatin receptor that allows for noninvasive gene transfer imaging. The purpose of this study was to identify the MTD, toxicities, clinical efficacy and biologic effects of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Experimental Design Eligible patients were treated intraperitoneally (IP) for 3 days with 1×109 to 1×1012 vp/dose of Ad5.SSTR/TK.RGD followed by intravenous ganciclovir for 14 days. Toxicity and clinical efficacy were assessed utilizing CTC Adverse Events grading and RECIST criteria. Imaging utilizing In-111 pentetreotide was obtained before and after treatment. Tissue samples were obtained to evaluate for gene transfer, generation of wild-type virus, viral shedding and antibody response. Results Twelve patients were treated in three cohorts. The most common vector-related clinical toxicities were grade 1–2 constitutional or pain symptoms, experienced most often in patients treated at the highest dose. MTD was not identified. Five patients demonstrated stable disease; all others experienced progressive disease. One patient with stable disease experienced complete resolution of disease and normalization of CA125 on further follow-up. Imaging detected increased In-111 pentetreotide retention in patients treated at the highest dose. Ancillary studies demonstrated presence of Ad5.SSTR/TK.RGD virus and HSV1-tk expression in ascites samples collected at various time points in most patients treated within the higher dose cohorts. Conclusions This study demonstrates the safety, potential efficacy, and possible gene transfer imaging capacity of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Further development of this novel gene therapeutic appears to be warranted. PMID:22510347

  16. [Adenovirus-delivered BMI-1 shRNA].

    PubMed

    Chen, Zhen-Ping; Chen, Xiao-Li; Zhen, Jie

    2009-10-01

    Recently, some plasmid vectors that direct transcription of small hairpin RNAs have been developed, which are processed into functional siRNAs by cellular enzymes. Although these vectors possess certain advantages over synthesized siRNA, many disadvantages exist, including low and variable transfection efficiency. This study was aimed to establish an adenoviral siRNA delivery system without above-mentioned disadvantages on the basis of commercially available vectors. A vector was designed to target the human polycomb gene BMI-1. The pAd-BMI-1shRNA-CMV-GFP vector was produced by cloning a 300 bp U6-BMI-1 cassette from the pGE1BMI-1shRNA plasmid and a CMV-GFP cassette from pAdTrack CMV in pShutter vector. The adenovirus was produced from the 293A packaging cell line and then infected K562 cells. The mRNA and protein levels of Bmi-1 were detected by real time-PCR and Western blot respectively. The results showed that the adenovirus carrying the BMI-1shRNA was successfully produced. After being transfected with the adenovirus, the K562 cells dramatically down-regulated BMI-1 expression, whereas the adenoviruses carrying control shRNA had no effect on BMI-1 expression. It is concluded that the adenoviruses are efficient vectors for delivery of siRNA into mammalian cells and may become a candidate vector carrying siRNA drugs for gene therapy. PMID:19840467

  17. Broad HIV Epitope Specificity and Viral Inhibition Induced by Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults

    PubMed Central

    Kopycinski, Jakub; Hayes, Peter; Ashraf, Ambreen; Cheeseman, Hannah; Lala, Francesco; Czyzewska-Khan, Justyna; Spentzou, Aggeliki; Gill, Dilbinder K.; Keefer, Michael C.; Excler, Jean-Louis; Fast, Patricia; Cox, Josephine; Gilmour, Jill

    2014-01-01

    A correlation between in vivo and in vitro virus control mediated by CD8+ T-cell populations has been demonstrated by CD8 T-cell-mediated inhibition of HIV-1 and SIV replication in vitro in peripheral blood mononuclear cells (PBMCs) from infected humans and non-human primates (NHPs), respectively. Here, the breadth and specificity of T-cell responses induced following vaccination with replication-defective adenovirus serotype 35 (Ad35) vectors containing a fusion protein of Gag, reverse transcriptase (RT), Integrase (Int) and Nef (Ad35-GRIN) and Env (Ad35-ENV), derived from HIV-1 subtype A isolates, was assessed in 25 individuals. The vaccine induced responses to a median of 4 epitopes per vaccinee. We correlated the CD8 responses to conserved vs. variable regions with the ability to inhibit a panel of 7 HIV-1 isolates representing multiple clades in a virus inhibition assay (VIA). The results indicate that targeting immunodominant responses to highly conserved regions of the HIV-1 proteome may result in an increased ability to inhibit multiple clades of HIV-1 in vitro. The data further validate the use of the VIA to screen and select future HIV vaccine candidates. Moreover, our data suggest that future T cell-focused vaccine design should aim to induce immunodominant responses to highly conserved regions of the virus. PMID:24609066

  18. Broad HIV epitope specificity and viral inhibition induced by multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

    PubMed

    Kopycinski, Jakub; Hayes, Peter; Ashraf, Ambreen; Cheeseman, Hannah; Lala, Francesco; Czyzewska-Khan, Justyna; Spentzou, Aggeliki; Gill, Dilbinder K; Keefer, Michael C; Excler, Jean-Louis; Fast, Patricia; Cox, Josephine; Gilmour, Jill

    2014-01-01

    A correlation between in vivo and in vitro virus control mediated by CD8+ T-cell populations has been demonstrated by CD8 T-cell-mediated inhibition of HIV-1 and SIV replication in vitro in peripheral blood mononuclear cells (PBMCs) from infected humans and non-human primates (NHPs), respectively. Here, the breadth and specificity of T-cell responses induced following vaccination with replication-defective adenovirus serotype 35 (Ad35) vectors containing a fusion protein of Gag, reverse transcriptase (RT), Integrase (Int) and Nef (Ad35-GRIN) and Env (Ad35-ENV), derived from HIV-1 subtype A isolates, was assessed in 25 individuals. The vaccine induced responses to a median of 4 epitopes per vaccinee. We correlated the CD8 responses to conserved vs. variable regions with the ability to inhibit a panel of 7 HIV-1 isolates representing multiple clades in a virus inhibition assay (VIA). The results indicate that targeting immunodominant responses to highly conserved regions of the HIV-1 proteome may result in an increased ability to inhibit multiple clades of HIV-1 in vitro. The data further validate the use of the VIA to screen and select future HIV vaccine candidates. Moreover, our data suggest that future T cell-focused vaccine design should aim to induce immunodominant responses to highly conserved regions of the virus. PMID:24609066

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

    Background We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. Methods Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×109 (A), 2×1010 (B), 2×1011 (C), or Ad35-GRIN 1×1010 (D) viral particles. Results No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A–D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 106 PBMC to any antigen was 78–139 across Groups A–C and 158–174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A–C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. Conclusion/Significance Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination

  1. Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate.

    PubMed

    Shen, Chun Fang; Jacob, Danielle; Zhu, Tao; Bernier, Alice; Shao, Zhongqi; Yu, Xuefeng; Patel, Mehul; Lanthier, Stephane; Kamen, Amine

    2016-06-17

    Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials. PMID:27154390

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

    PubMed Central

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

    1971-01-01

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

  3. Genomic and Bioinformatics Analysis of HAdV-4, a Human Adenovirus Causing Acute Respiratory Disease: Implications for Gene Therapy and Vaccine Vector Development

    PubMed Central

    Purkayastha, Anjan; Ditty, Susan E.; Su, Jing; McGraw, John; Hadfield, Ted L.; Tibbetts, Clark; Seto, Donald

    2005-01-01

    Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD. PMID:15681456

  4. Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development.

    PubMed

    Purkayastha, Anjan; Ditty, Susan E; Su, Jing; McGraw, John; Hadfield, Ted L; Tibbetts, Clark; Seto, Donald

    2005-02-01

    Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD. PMID:15681456

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Adenovirus Vector Expressing Stx1/Stx2-Neutralizing Agent Protects Piglets Infected with Escherichia coli O157:H7 against Fatal Systemic Intoxication

    PubMed Central

    Sheoran, Abhineet S.; Dmitriev, Igor P.; Kashentseva, Elena A.; Cohen, Ocean; Mukherjee, Jean; Debatis, Michelle; Shearer, Jonathan; Tremblay, Jacqueline M.; Beamer, Gillian; Curiel, David T.; Shoemaker, Charles B.

    2014-01-01

    Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only VH domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC. PMID:25368111

  7. Adenovirus vector expressing Stx1/Stx2-neutralizing agent protects piglets infected with Escherichia coli O157:H7 against fatal systemic intoxication.

    PubMed

    Sheoran, Abhineet S; Dmitriev, Igor P; Kashentseva, Elena A; Cohen, Ocean; Mukherjee, Jean; Debatis, Michelle; Shearer, Jonathan; Tremblay, Jacqueline M; Beamer, Gillian; Curiel, David T; Shoemaker, Charles B; Tzipori, Saul

    2015-01-01

    Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only VH domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC. PMID:25368111

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Vijayalingam, S; Kuppuswamy, M; Subramanian, T; Strebeck, F F; West, C L; Varvares, M; Chinnadurai, G

    2014-06-01

    Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9,10-dimethyl-1,2-benzanthracene. These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55 K (which are defective in the expression of either E1B-19 K alone or both E1B-19 K and E1B-55 K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intratumoral administration of HAdV5-E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting that virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers. PMID:24874842

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

    PubMed Central

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

    2015-01-01

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

  11. Adenovirus receptors and their implications in gene delivery

    PubMed Central

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

    2010-01-01

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

  12. The Arg279Glu Substitution in the Adenovirus Type 11p (Ad11p) Fiber Knob Abolishes EDTA-Resistant Binding to A549 and CHO-CD46 Cells, Converting the Phenotype to That of Ad7p

    PubMed Central

    Gustafsson, Dan J.; Segerman, Anna; Lindman, Kristina; Mei, Ya-Fang; Wadell, Göran

    2006-01-01

    The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Glu substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46. PMID:16439545

  13. Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform.

    PubMed

    Gabitzsch, Elizabeth S; Balint-Junior, Joseph P; Xu, Younong; Balcaitis, Stephanie; Sanders-Beer, Brigitte; Karl, Julie; Weinhold, Kent J; Paessler, Slobodan; Jones, Frank R

    2012-11-26

    Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform. PMID:23041546

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

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

    2015-08-15

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

  16. In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

    PubMed Central

    Lusky, M.; Christ, M.; Rittner, K.; Dieterle, A.; Dreyer, D.; Mourot, B.; Schultz, H.; Stoeckel, F.; Pavirani, A.; Mehtali, M.

    1998-01-01

    Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome. PMID:9499056

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

    ERIC Educational Resources Information Center

    Heckler, Andrew F.; Scaife, Thomas M.

    2015-01-01

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

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

    PubMed

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

    2007-07-01

    Genetic deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, also known as von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia and growth retardation. We tested whether helper-dependent adenovirus (HDAd)-mediated hepatic delivery of G6Pase would lead to prolonged survival and sustained correction of the metabolic abnormalities in G6Pase knockout (KO) mice, a model for a severe form of GSD-Ia. An HDAd vector encoding G6Pase was administered intravenously (2 or 5 x 10(12)vector particles/kg) to 2-week-old (w.o.) G6Pase-KO mice. Following HDAd vector administration survival was prolonged to a median of 7 months, in contrast to untreated affected mice that did not survive past 3 weeks of age. G6Pase levels increased more than tenfold between 3 days and 28 weeks after HDAd injection (P < 0.03). The weights of untreated 2 w.o. G6Pase-KO mice were approximately half those of their unaffected littermates, and treatment stimulated their growth to the size of wild-type mice. Severe hypoglycemia and hypercholesterolemia, which are hallmarks of GSD-Ia both in humans and in mice, were also restored to normalcy by the treatment. Glycogen accumulation in the liver was markedly reduced. The efficacy of HDAd-G6Pase treatment in reversing the physiological and biochemical abnormalities associated with GSD-Ia in affected G6Pase-KO mice justifies further preclinical evaluation in murine and canine models of GSD-Ia. PMID:17505475

  19. Evaluation of novel large cut-off ultrafiltration membranes for adenovirus serotype 5 (Ad5) concentration.

    PubMed

    Nestola, Piergiuseppe; Martins, Duarte L; Peixoto, Cristina; Roederstein, Susanne; Schleuss, Tobias; Alves, Paula M; Mota, José P B; Carrondo, Manuel J T

    2014-01-01

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

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

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

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

  1. Protective role of adenovirus vector-mediated interleukin-10 gene therapy on endogenous islet β-cells in recent-onset type 1 diabetes in NOD mice

    PubMed Central

    LI, CHENG; ZHANG, LIJUAN; CHEN, YANYAN; LIN, XIAOJIE; LI, TANG

    2016-01-01

    The aim of the present study was to provide an animal experimental basis for the protective effect of the adenoviral vector-mediated interleukin-10 (Ad-mIL-10) gene on islet β-cells during the early stages of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. A total of 24 female NOD mice at the onset of diabetes were allocated at random into three groups (n=8 per group): Group 1, intraperitoneally injected with 0.1 ml Ad-mIL-10; group 2, intraperitoneally injected with 0.1 ml adenovirus vector; and group 3, was a diabetic control. In addition to groups 1, 2 and 3, 8 age- and gender-matched NOD mice were intraperitoneally injected with 0.1 ml PBS and assigned to group 4 as a normal control. All mice were examined weekly for body weight, urine glucose and blood glucose values prior to onset of diabetes, and at 1, 2 and 3 weeks after that, and all mice were sacrificed 3 weeks after injection. Serum levels of interleukin (IL)-10, interferon (IFN)-γ, IL-4, insulin and C-peptide were evaluated, and in addition the degree of insulitis and the local expression of IL-10 gene in the pancreas were detected. The apoptosis rate of pancreatic β-cells was determined using a TUNEL assay. Compared with groups 2 and 3, IL-10 levels in the serum and pancreas were elevated in group 1. Serum IFN-γ levels were decreased while serum IL-4 levels and IFN-γ/IL-4 ratio were significantly increased in group 1 (P<0.01). C-peptide and insulin levels were higher in group 1 compared with groups 2 and 3, (P<0.01). Furthermore, compared with groups 2 and 3, the degree of insulitis, islet β-cell apoptosis rate and blood glucose values did not change significantly (P>0.05). The administration of the Ad-mIL-10 gene induced limited immune regulatory and protective effects on islet β-cell function in NOD mice with early T1D, while no significant reduction in insulitis, islet β-cell apoptosis rate and blood glucose was observed. PMID:27168782

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

    PubMed

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

    2008-10-01

    Rabies infections in swine have been reported occasionally in recent years in certain geographic locations. Although a protective vaccine consisting of inactivated rabies virus is available for use in swine, searching for a more economically viable formulation for use in developing countries is always a priority. This work describes the testing of a canine adenovirus that expresses a rabies viral epitope (CAV-2-E3Delta-RGP) in a porcine rabies model. The data presented here show that the recombinant viral vaccine was effective in protecting swine against rabies if administered intramuscularly, but not orally or intranasally, and that protection was probably related to the development of a humoral response that lasted at least 28 weeks. Following vaccination, no behavioral abnormalities were observed in vaccinated swine and virus particles were not detected in either tissues or body fluids, indicating that this formulation was safe. The recombinant virus stimulated an effective level of antibody response in the immunized swine after a single intramuscular inoculation. PMID:18721839

  3. Modulation of Treg function improves adenovirus vector-mediated gene expression in the airway.

    PubMed

    Nagai, Y; Limberis, M P; Zhang, H

    2014-02-01

    Virus vector-mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the cystic fibrosis transmembrane conductance regulator gene. The pathological proinflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limits the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (T(reg)) and to improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway. PMID:24385144

  4. Innate Immunity to Adenovirus

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2011-05-01

    Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response. PMID:21190734

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Heckler, Andrew F.; Scaife, Thomas M.

    2015-06-01

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

  10. Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes.

    PubMed

    Molinier-Frenkel, V; Gahery-Segard, H; Mehtali, M; Le Boulaire, C; Ribault, S; Boulanger, P; Tursz, T; Guillet, J G; Farace, F

    2000-08-01

    We previously demonstrated that a single injection of 10(9) PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218-2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8(+) CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

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

    PubMed Central

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

    2000-01-01

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

  12. Vaccination with Adenovirus Serotypes 35, 26, and 48 Elicits Higher Levels of Innate Cytokine Responses than Adenovirus Serotype 5 in Rhesus Monkeys

    PubMed Central

    Teigler, Jeffrey E.; Iampietro, M. Justin

    2012-01-01

    Adenovirus (Ad) vaccine vectors have proven highly immunogenic in multiple experimental models, but the innate immune responses induced by these vectors remain poorly characterized. Here we report innate cytokine responses to 5 different Ad vectors in 26 rhesus monkeys. Vaccination with adenovirus serotype 35 (Ad35), Ad26, and Ad48 induced substantially higher levels of antiviral (gamma interferon [IFN-γ], 10-kDa gamma interferon-induced protein [IP-10]) and proinflammatory (interleukin 1 receptor antagonist [IL-1RA], IL-6) cytokines than vaccination with Ad5 on day 1 following immunization. In vitro studies with capsid chimeric vectors and receptor-blocking monoclonal antibodies suggested that fiber-receptor interactions, as well as other capsid components, were critical for triggering these innate responses. Moreover, multiple cell populations, including dendritic cells, monocytes/macrophages, and T lymphocytes, contributed to these innate cytokine profiles. These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their primary cellular receptor, induce significantly greater innate cytokine responses than Ad5, which uses the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors. PMID:22787208

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

    PubMed Central

    2011-01-01

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

  14. AN AD5-SIV VACCINE FOR PROTECTION AGAINST SWINE FLU (H3N2)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant human adenovirus (rAd) vectored vaccines for pseudorabies (PRV) have been shown to prime immunity in neonatal piglets and to overcome interference by maternal-derived anti-PRV antibodies (J. Gen. Virol. 78:3303-10, 1997). Similarly, replication-incompetent adenoviruses containing either...

  15. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer.

    PubMed

    Walters, R; Welsh, M

    1999-11-01

    Delivery of a normal copy of CFTR cDNA to airway epithelia may provide a novel treatment for cystic fibrosis lung disease. Unfortunately, current vectors are inefficient because of limited binding to the apical surface of airway epithelia. We recently reported that incorporation of adenovirus in a calcium phosphate coprecipitate (Ad:CaPi) improves adenovirus-mediated gene transfer to airway epithelia in vitro and in vivo. To understand better how coprecipitation improves gene transfer, we tested the hypothesis that incorporation in a CaPi coprecipitate increases the binding of adenovirus to the apical surface of differentiated human airway epithelia. When a Cy3-labelled adenovirus was delivered in a coprecipitate, binding increased 54-fold as compared with adenovirus alone. Moreover, infection by Ad:CaPi was independent of fiber knob-CAR and penton base-integrin interactions. After binding to the cell surface, the virus must enter the cell in order to infect. We hypothesized that Ad:CaPi may stimulate fluid phase endocytosis, thereby facilitating entry. However, we found that neither adenovirus nor Ad:CaPi coprecipitates altered fluid phase endocytosis. Nevertheless, Ad:CaPi preferentially infected cells showing endocytosis. Thus, CaPi coprecipitation improves adenovirus-mediated gene transfer by coating the epithelial surface with a layer of virus which enters cells during the normal process of endocytosis. PMID:10602380

  16. Protection Against Chikungunya Virus Induced Arthralgia Following Prophylactic Treatment with Adenovirus Vectored Interferon (mDEF201)

    PubMed Central

    Dagley, Ashley; Ennis, Jane; Turner, Jeffrey D.; Rood, Kerry A.; Van Wettere, Arnaud J.; Gowen, Brian B.; Julander, Justin G.

    2014-01-01

    Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ~100-fold while spleen virus increased >1,000-fold within 1 day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7 dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5 dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 107 pfu/ml dose of mDEF201 administered 21 days to 24 h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24 h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease. PMID:24833276

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

    PubMed Central

    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

  18. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.

    PubMed

    Gordon, J W

    2001-04-01

    The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10(10) infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 x 10(8) particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adbeta-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5-7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low. PMID:11319918

  19. Neogenesis and proliferation of {beta}-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    SciTech Connect

    Tokui, Yae . E-mail: ytokui@imed2.med.osaka-u.ac.jp; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro |

    2006-12-01

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

  20. 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

    PubMed Central

    Hayton, Emma-Jo; Rose, Annie; Ibrahimsa, Umar; Del Sorbo, Mariarosaria; Capone, Stefania; Crook, Alison; Black, Antony P.; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    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 safety and good

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

    SciTech Connect

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

    2014-10-15

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

  2. Polymeric oncolytic adenovirus for cancer gene therapy

    PubMed Central

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

    2015-01-01

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

  3. Gene Delivery by Subconjunctival Injection of Adenovirus in Rats: A Study of Local Distribution, Transgene Duration and Safety

    PubMed Central

    Lee, Jia Hui; Tsai, Pei-Jhen; Tsai, Han-En; Sheu, Shwu-Jiuan; Lin, Hsiu-Chen; Dusting, Gregory J.; Tai, Ming-Hong; Bee, Youn-Shen

    2015-01-01

    Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, β-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2014-02-15

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

  6. Cellular Changes Induced by Adenovirus Vaccine Vectors Expressing Foot-and-Mouth Disease Virus Structural and Nonstructural Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) is the most contagious pathogen of cloven-hoofed animals including swine and bovines. The emergency control of outbreaks is dependent on rapid protection and prevention of virus spread. Adenovirus-based FMD subunit vaccines containing the coding region of viral ca...

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2012-04-01

    Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia. PMID:21654821

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  11. Immunogenicity and efficacy of the novel cancer vaccine based on simian adenovirus and MVA vectors alone and in combination with PD-1 mAb in a mouse model of prostate cancer.

    PubMed

    Cappuccini, Federica; Stribbling, Stephen; Pollock, Emily; Hill, Adrian V S; Redchenko, Irina

    2016-06-01

    Prostate cancer possesses several characteristics that make it a suitable candidate for immunotherapy; however, prostate cancer vaccines to date demonstrate modest efficacy and low immunogenicity. The goal of the present pre-clinical study was to explore the immunogenic properties and protective efficacy of a novel prostate cancer immunotherapy based on the heterologous prime-boost viral-vectored vaccination platform. The simian adenovirus, ChAdOx1, and modified vaccinia Ankara virus, MVA, encoding a prostate cancer-associated antigen, the six transmembrane epithelial antigen of the prostate 1 (STEAP1), induced strong sustained antigen-specific CD8+ T-cell responses in C57BL/6 and BALB/c male mice. Unexpectedly, the high vaccine immunogenicity translated into relatively low protective efficacy in the murine transplantable and spontaneous models of prostate cancer. A combination of the vaccine with PD-1 blocking antibody significantly improved survival of the animals, with 80 % of mice remaining tumour-free. These results indicate that the ChAdOx1-MVA vaccination regime targeting STEAP1 combined with PD-1 therapy might have high therapeutic potential in the clinic. PMID:27052571

  12. Pre-Existing Adenovirus Immunity Modifies a Complex Mixed Th1 and Th2 Cytokine Response to an Ad5/HIV-1 Vaccine Candidate in Humans

    PubMed Central

    Pine, Samuel O.; Kublin, James G.; Hammer, Scott M.; Borgerding, Joleen; Huang, Yunda; Casimiro, Danilo R.; McElrath, M. Juliana

    2011-01-01

    The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest

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

    PubMed

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

    2012-01-01

    Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity. PMID:22514686

  14. Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses.

    PubMed

    Holman, David H; Wang, Danher; Raja, Nicholas U; Luo, Min; Moore, Kevin M; Woraratanadharm, Jan; Mytle, Nutan; Dong, John Y

    2008-05-19

    There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine. PMID:18395306

  15. In vitro CRISPR-Cas9-mediated efficient Ad5 vector modification.

    PubMed

    Tang, Lichun; Gong, Mengmeng; Zhang, Pumin

    2016-05-27

    The CRISPR-Cas9 genome editing system has been widely used in multiple cells and organisms. Here we developed a CRISPR-Cas9 based in vitro large DNA vector editing system, using the Ad5-based vector as an example. We demonstrate use of this system to generate targeted mutations, in-frame gene deletion, and gene replacement. This in vitro CRISPR editing system exhibits high efficiency and accuracy. We believe this system can be applied in a variety of experimental settings. PMID:27125457

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

    SciTech Connect

    Banerjee, Rabin Majhi, Bibhas Ranjan

    2008-03-15

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

  17. Characterization of infectivity of knob-modified adenoviral vectors in glioma.

    PubMed

    Paul, C P L; Everts, M; Glasgow, J N; Dent, P; Fisher, P B; Ulasov, I V; Lesniak, M S; Stoff-Khalili, M A; Roth, J C; Preuss, M A; Dirven, C M F; Lamfers, M L M; Siegal, G P; Zhu, Z B; Curiel, D T

    2008-05-01

    Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma. PMID:18756624

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

    PubMed Central

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

    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 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. PMID:25368113

  19. [The construction of recombinant adenovirus expressing bifunctional fusion protein sCAR-EGF and the detection of its activity].

    PubMed

    Ren, Peng-Kang; Wang, Feng; Li, Hui-Ming; Li, Zong-Hai; Huang, Qian

    2006-09-01

    To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR. PMID:17037191

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

    NASA Astrophysics Data System (ADS)

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

    2009-04-01

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

  1. mAbs and Ad-vectored IFN-α therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms.

    PubMed

    Qiu, Xiangguo; Wong, Gary; Fernando, Lisa; Audet, Jonathan; Bello, Alexander; Strong, Jim; Alimonti, Judie B; Kobinger, Gary P

    2013-10-16

    ZMAb is a promising treatment against Ebola virus (EBOV) disease that has been shown to protect 50% (two of four) of nonhuman primates (NHPs) when administered 2 days post-infection (dpi). To extend the treatment window and improve protection, we combined ZMAb with adenovirus-vectored interferon-α (Ad-IFN) and evaluated efficacy in EBOV-infected NHPs. Seventy-five percent (three of four) and 100% (four of four) of cynomolgus and rhesus macaques survived, respectively, when treatment was initiated after detection of viremia at 3 dpi. Fifty percent (two of four) of the cynomolgus macaques survived when Ad-IFN was given at 1 dpi, followed by ZMAb starting at 4 dpi, after positive diagnosis. The treatment was able to suppress viremia reaching ~10(5) TCID50 (median tissue culture infectious dose) per milliliter, leading to survival and robust specific immune responses. This study describes conditions capable of saving 100% of EBOV-infected NHPs when initiated after the presence of detectable viremia along with symptoms. PMID:24132638

  2. Evaluation of the Efficacy of ChAd63-MVA Vectored Vaccines Expressing Circumsporozoite Protein and ME-TRAP Against Controlled Human Malaria Infection in Malaria-Naive Individuals

    PubMed Central

    Hodgson, Susanne H.; Ewer, Katie J.; Bliss, Carly M.; Edwards, Nick J.; Rampling, Thomas; Anagnostou, Nicholas A.; de Barra, Eoghan; Havelock, Tom; Bowyer, Georgina; Poulton, Ian D.; de Cassan, Simone; Longley, Rhea; Illingworth, Joseph J.; Douglas, Alexander D.; Mange, Pooja B.; Collins, Katharine A.; Roberts, Rachel; Gerry, Stephen; Berrie, Eleanor; Moyle, Sarah; Colloca, Stefano; Cortese, Riccardo; Sinden, Robert E.; Gilbert, Sarah C.; Bejon, Philip; Lawrie, Alison M.; Nicosia, Alfredo; Faust, Saul N.; Hill, Adrian V. S.

    2015-01-01

    Background. Circumsporozoite protein (CS) is the antigenic target for RTS,S, the most advanced malaria vaccine to date. Heterologous prime-boost with the viral vectors simian adenovirus 63 (ChAd63)-modified vaccinia virus Ankara (MVA) is the most potent inducer of T-cells in humans, demonstrating significant efficacy when expressing the preerythrocytic antigen insert multiple epitope–thrombospondin-related adhesion protein (ME-TRAP). We hypothesized that ChAd63-MVA containing CS may result in a significant clinical protective efficacy. Methods. We conducted an open-label, 2-site, partially randomized Plasmodium falciparum sporozoite controlled human malaria infection (CHMI) study to compare the clinical efficacy of ChAd63-MVA CS with ChAd63-MVA ME-TRAP. Results. One of 15 vaccinees (7%) receiving ChAd63-MVA CS and 2 of 15 (13%) receiving ChAd63-MVA ME-TRAP achieved sterile protection after CHMI. Three of 15 vaccinees (20%) receiving ChAd63-MVA CS and 5 of 15 (33%) receiving ChAd63-MVA ME-TRAP demonstrated a delay in time to treatment, compared with unvaccinated controls. In quantitative polymerase chain reaction analyses, ChAd63-MVA CS was estimated to reduce the liver parasite burden by 69%–79%, compared with 79%–84% for ChAd63-MVA ME-TRAP. Conclusions. ChAd63-MVA CS does reduce the liver parasite burden, but ChAd63-MVA ME-TRAP remains the most promising antigenic insert for a vectored liver-stage vaccine. Detailed analyses of parasite kinetics may allow detection of smaller but biologically important differences in vaccine efficacy that can influence future vaccine development. Clinical Trials Registration. NCT01623557. PMID:25336730

  3. [Preparation of monoclonal antibodies against enterovirus type 71 with an epitope-incorporated adenovirus type 3 vector].

    PubMed

    Fan, Ye; Tian, Xingui; Xue, Chunyan; Liu, Minglong; Zhou, Zhichao; Li, Xiao; Li, Chenyang; Zhou, Rong

    2016-08-01

    Objective To develop the monoclonal antibodies (mAbs) against enterovirus type 71 (EV71). Methods Two neutralization epitopes, SP70 and SP55, from EV71 were cloned into the hexon gene of adenovirus type 3 to generate a recombinant adenovirus type 3 (R1R2A3) presenting SP70 and SP55 antigens. BALB/c mice were immunized with the R1R2A3. The mAbs were developed with hybridoma technology and were analyzed with microneutralizing assay, indirect ELISA, Western blotting and direct immunofluorescence assay (DFA). Results The study obtained four hybridoma cell clones, 2C4, D2C9, I2G2 and I12C3. ELISA showed that the titer of D2C9 against EV71 was 1:8 000 000 and the titers of 2C4, I2G2, and I12C3 all were 1:500 000. ELISA and Western blotting demonstrated that all mAbs could specifically recognize the VP1 of EV71. In addition, D2C9 recognized the SP70 epitope, and 2C4, I12C3 and I2G2 all recognized the SP55 epitope. DFA revealed that all mAbs could react with EV71, but not with Coxsackie virus A16 (CoxA16). Conclusion Four mAbs against EV71 have been developed successfully, and all of them could react with EV71 rather than CoxA16. PMID:27412945

  4. [Generation and preliminary immunological efficacy of a recombinant human adenovirus-rabies virus glycoprotein].

    PubMed

    Wang, Ying; Zhang, Shou-Feng; Liu, Ye; Zhang, Fei; Zhang, Jin-Xia; Hu, Rong-Liang

    2011-09-01

    To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine. PMID:21998956

  5. [The complexes of adenovirus and anionic liposomes: preparation and in vitro characterization].

    PubMed

    Zhong, Zhi-Rong; Wan, Yu; Shi, San-Jun; Zhang, Zhi-Rong; Sun, Xun

    2012-01-01

    This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5. PMID:22493816

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-02-01

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

  8. A fiber-modified adenovirus co-expressing HSV-TK and Coli.NTR enhances antitumor activities in breast cancer cells

    PubMed Central

    Zhan, Yang; Yu, Bin; Wang, Zhen; Zhang, Yu; Zhang, Hai-Hong; Wu, Hao; Feng, Xiao; Geng, Ran-Shen; Kong, Wei; Yu, Xiang-Hui

    2014-01-01

    Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer. PMID:25031704

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Canine adenovirus downstream processing protocol.

    PubMed

    Puig, Meritxell; Piedra, Jose; Miravet, Susana; Segura, María Mercedes

    2014-01-01

    Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %. PMID:24132487

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

    PubMed

    Lin, Shih-Chang; Liu, Wen-Chun; Jan, Jia-Tsrong; Wu, Suh-Chin

    2014-01-01

    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. PMID:24671139

  14. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

    PubMed

    Stanley, Daphne A; Honko, Anna N; Asiedu, Clement; Trefry, John C; Lau-Kilby, Annie W; Johnson, Joshua C; Hensley, Lisa; Ammendola, Virginia; Abbate, Adele; Grazioli, Fabiana; Foulds, Kathryn E; Cheng, Cheng; Wang, Lingshu; Donaldson, Mitzi M; Colloca, Stefano; Folgori, Antonella; Roederer, Mario; Nabel, Gary J; Mascola, John; Nicosia, Alfredo; Cortese, Riccardo; Koup, Richard A; Sullivan, Nancy J

    2014-10-01

    Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge. PMID:25194571

  15. Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

    PubMed Central

    Damjanovic, Daniela; Zhang, Xizhong; Mu, Jingyu; Fe Medina, Maria; Xing, Zhou

    2008-01-01

    It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern. PMID:18261231

  16. Genomic and bioinformatics analysis of HAdV-7, a human adenovirus of species B1 that causes acute respiratory disease: implications for vector development in human gene therapy.

    PubMed

    Purkayastha, Anjan; Su, Jing; Carlisle, Steve; Tibbetts, Clark; Seto, Donald

    2005-02-01

    Human adenovirus serotype 7 (HAdV-7) is a reemerging pathogen identified in acute respiratory disease (ARD), particularly in epidemics affecting basic military trainee populations of otherwise healthy young adults. The genome has been sequenced and annotated (GenBank accession no. ). Comparative genomics and bioinformatics analyses of the HAdV-7 genome sequence provide insight into its natural history and phylogenetic relationships. A putative origin of HAdV-7 from a chimpanzee host is observed. This has implications within the current biotechnological interest of using chimpanzee adenoviruses as vectors for human gene therapy and DNA vaccine delivery. Rapid genome sequencing and analyses of this species B1 member provide an example of exploiting accurate low-pass DNA sequencing technology in pathogen characterization and epidemic outbreak surveillance through the identification, validation, and application of unique pathogen genome signatures. PMID:15661145

  17. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

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

  18. [Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A].

    PubMed

    Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Xu, Yi; Jia, Run-Qing; Bo, Hong; Dong, Jie; Zhou, Jian-Fang; Shu, Yue-Long; Zhang, Zhi-Qing

    2009-03-01

    Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector. PMID:19678564

  19. A Single Intraduodenal Administration of Human Adenovirus 40 Vaccine Effectively Prevents Anaphylactic Shock

    PubMed Central

    Yamasaki, Satoshi; Miura, Yoshiaki; Davydova, Julia; Vickers, Selwyn M.

    2013-01-01

    Vaccine administration into the intestine is known to induce mucosal tolerance most efficiently. Therefore, developing a delivery system that targets the intestinal mucosa is expected to improve the efficiency of immunosuppression. Human enteric adenovirus serotype 40 (Ad40)-based vectors have the advantage of targeting intestinal mucosa, making them prime candidates as mucosal vaccine carriers for immunosuppression. Here, after both oral and intraduodenal administrations, the vector distribution of replication-defective recombinant Ad40 vectors (rAd40) was significantly higher than that of a conventional Ad vector based on human adenovirus 5 (Ad5) in ilea containing Peyer's patches. Single intraduodenal administration of rAd40 induced antigen-specific mucosal immunoreaction mediated by intestinal mucosal and systemic immunity. In ovalbumin-induced allergy mouse models, this approach inhibited antigen-specific delayed-type hypersensitivity reactions, diarrhea occurrence, and systemic anaphylaxis. Thus, a single intraduodenal administration of rAd40 provides a potent method of inducing allergen-specific mucosal tolerance and a new allergen-specific immunotherapy for overcoming problems with current therapies against life-threatening allergic reactions, including anaphylaxis. PMID:23885027

  20. [Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].

    PubMed

    Zou, Xiaohui; Xiao, Rong; Guo, Xiaojuan; Qu, Jianguo; Lu, Zhuozhuang; Hong, Tao

    2016-01-01

    We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM. PMID:27295881

  1. The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

    PubMed Central

    Pantelidou, Constantia; Cherubini, Gioia; Lemoine, Nick R.; Halldén, Gunnel

    2016-01-01

    Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics. PMID:26872382

  2. The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11.

    PubMed

    Pantelidou, Constantia; Cherubini, Gioia; Lemoine, Nick R; Halldén, Gunnel

    2016-03-29

    Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics. PMID:26872382

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

    PubMed

    Schutta, Christopher; Barrera, José; Pisano, Melia; Zsak, Laszlo; Grubman, Marvin J; Mayr, Gregory A; Moraes, Mauro P; Kamicker, Barbara J; Brake, David A; Ettyreddy, Damodar; Brough, Douglas E; Butman, Bryan T; Neilan, John G

    2016-06-01

    The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge. PMID:26707216

  4. Adenovirus Vector-Induced CD8+ T Effector Memory Cell Differentiation and Recirculation, But Not Proliferation, Are Important for Protective Immunity Against Experimental Trypanosoma cruzi Infection

    PubMed Central

    Vasconcelos, José Ronnie; Dominguez, Mariana R.; Neves, Ramon L.; Ersching, Jonatan; Araújo, Adriano; Santos, Luara I.; Virgilio, Fernando S.; Machado, Alexandre V.; Bruna-Romero, Oscar; Gazzinelli, Ricardo T.

    2014-01-01

    Abstract Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8+ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8+ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1High CD27Low CD43Low CD183LowT-betHigh EomesLow phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8+ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8+ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8+ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity. PMID:24568548

  5. Adenovirus-Mediated Expression of Interferon-Alpha Delays Viral Replication and Reduces Disease Signs in Swine Challenged with Porcine Reproductive and Respiratory Syndrome Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, pigs were injected with a nonreplicating human adenovirus type 5 vector expressing porcine interferon-alpha (Ad5-pIFNa) and then challenged with porcine reproductive and respiratory syndrome virus (PRRSV) to determine whether the presence of increased levels of IFNa would decrease vir...

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

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases. PMID:26467629

  7. Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2).

    PubMed

    Tang, M; Harp, J A; Wesley, R D

    2002-11-01

    Immunization with recombinant adenoviral vaccine that induces potent immunity has been applied to many infectious diseases. We report here developing a recombinant adenoviral vaccine encoding the HA gene from swine H3N2 influenza virus (SIV). Two replication-defective recombinant adenoviruses were generated: (1) rAd-HA: recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus, and (2) rAd-vector: a control recombinant adenovirus containing adenovirus and transfer plasmids without a foreign HA gene. Mice given rAd-HA developed high titers of neutralizing and hemagglutination inhibition antibodies to SIV in comparison to mice inoculated with rAd-vector or PBS as early as 2 weeks after immunization, and these antibodies were substantially increased in the mice given rAd-HA within the next 3 weeks following the first dose. However, these antibodies were not able to neutralize the virus, A/HK/68 (H3N2), used for challenge. Nonetheless mice immunized with one or two doses of rAd-HA were protected from lethal challenge with heterologous virus, A/HK/1/68 (H3N2). A statistically significant ( P < 0.03) difference between survival rates of rAd-HA mice vs. rAd-vector or PBS mice was observed. PMID:12417948

  8. Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents

    SciTech Connect

    Shashkova, Elena V.; May, Shannon M.; Barry, Michael A.

    2009-11-25

    Human adenovirus type 5 (Ad5) has been the most popular platform for the development of oncolytic Ads. Alternative Ad serotypes with low seroprevalence might allow for improved anticancer efficacy in Ad5-immune patients. We studied the safety and efficacy of rare serotypes Ad6, Ad11 and Ad35. In vitro cytotoxicity of the Ads correlated with expression of CAR and CD46 in most but not all cell lines. Among CAR-binding viruses, Ad5 was often more active than Ad6, among CD46-binding viruses Ad35 was generally more cytotoxic than Ad11 in cell culture studies. Ad5, Ad6, and Ad11 demonstrated similar anticancer activity in vivo, whereas Ad35 was not efficacious. Hepatotoxicity developed only in Ad5-injected mice. Predosing with Ad11 and Ad35 did not increase infection of hepatocytes with Ad5-based vector demonstrating different interaction of these Ads with Kupffer cells. Data obtained in this study suggest developing Ad6 and Ad11 as alternative Ads for anticancer treatment.

  9. Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

    PubMed

    Xia, Shui-Li; Lei, Jian-Lin; Du, Mingliang; Wang, Yimin; Cong, Xin; Xiang, Guang-Tao; Li, Lian-Feng; Yu, Shenye; Du, Enqi; Liu, Siguo; Sun, Yuan; Qiu, Hua-Ji

    2016-01-01

    Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines. PMID:27301745

  10. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  11. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

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

  12. Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure

    PubMed Central

    Wirtz, S; Galle, P; Neurath, M

    1999-01-01

    standard AdCMVβGal vectors.
CONCLUSIONS—Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.


Keywords: adenovirus; gene transfer; colitis; colon PMID:10323880

  13. Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.

    PubMed

    Barouch, Dan H; Alter, Galit; Broge, Thomas; Linde, Caitlyn; Ackerman, Margaret E; Brown, Eric P; Borducchi, Erica N; Smith, Kaitlin M; Nkolola, Joseph P; Liu, Jinyan; Shields, Jennifer; Parenteau, Lily; Whitney, James B; Abbink, Peter; Ng'ang'a, David M; Seaman, Michael S; Lavine, Christy L; Perry, James R; Li, Wenjun; Colantonio, Arnaud D; Lewis, Mark G; Chen, Bing; Wenschuh, Holger; Reimer, Ulf; Piatak, Michael; Lifson, Jeffrey D; Handley, Scott A; Virgin, Herbert W; Koutsoukos, Marguerite; Lorin, Clarisse; Voss, Gerald; Weijtens, Mo; Pau, Maria G; Schuitemaker, Hanneke

    2015-07-17

    Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys. PMID:26138104

  14. Protective Efficacy of Adenovirus/Protein Vaccines Against SIV Challenges in Rhesus Monkeys

    PubMed Central

    Barouch, Dan H.; Alter, Galit; Broge, Thomas; Linde, Caitlyn; Ackerman, Margaret E.; Brown, Eric P.; Borducchi, Erica N.; Smith, Kaitlin M.; Nkolola, Joseph P.; Liu, Jinyan; Shields, Jennifer; Parenteau, Lily; Whitney, James B.; Abbink, Peter; Ng’ang’a, David M.; Seaman, Michael S.; Lavine, Christy L.; Perry, James R.; Li, Wenjun; Colantonio, Arnaud D.; Lewis, Mark G.; Chen, Bing; Wenschuh, Holger; Reimer, Ulf; Piatak, Michael; Lifson, Jeffrey D.; Handley, Scott A.; Virgin, Herbert W.; Koutsoukos, Marguerite; Lorin, Clarisse; Voss, Gerald; Weijtens, Mo; Pau, Maria G.; Schuitemaker, Hanneke

    2015-01-01

    Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against stringent virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by boosting with a purified envelope (Env) glycoprotein. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env/Gag/Pol antigens and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repetitive, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repetitive, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of stringent virus challenges in rhesus monkeys. PMID:26138104

  15. Strain-dependent and distinctive T-cell responses to HIV antigens following immunisation of mice with differing chimpanzee adenovirus vaccine vectors.

    PubMed

    Herath, S; Le Heron, A; Colloca, S; Patterson, S; Tatoud, R; Weber, J; Dickson, G

    2016-08-17

    In vivo vaccination studies are conventionally conducted in a single mouse strain with results, only reflecting responses to a single immunogenetic background. We decided to examine the immune response to an HIV transgene (gag, pol and nef fusion protein) in 3 strains of mice (CBA, C57BL/6 and BALB/c) to determine the spectrum of responses and in addition to determine whether the serotype of the adenoviral vector used (ChAd3 and ChAd63) impacted the outcome of response. Our results demonstrated that all three strains of mice responded to the transgene and that the magnitude of responses were different between the strains. The C57BL/6 strain showed the lowest range of responses compared to the other strains and, very few responses were seen to the same peptide pool in all three strains of mice. In CBA and BALB/c mice there were significant differences in IFNγ production dependent on the adenoviral vector used. Our results suggest that employing a single strain of mouse may underestimate the efficacy and efficiency of vaccine products. PMID:27452864

  16. Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression

    PubMed Central

    Stilwell, Jackie L.; McCarty, Douglas M.; Negishi, Atsuko; Superfine, Richard; Samulski, R. Jude

    2003-01-01

    Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone. PMID:14610209

  17. [Characteristics of intranuclear inclusions formed during the reproduction of bovine adenoviruses].

    PubMed

    Nosach, L N; Belousova, R V; Diachenko, N S; Kolenkova, L M

    1986-01-01

    A cytomorphological method was used to study the reproduction of bovine adenoviruses: Ad bos 1 - Ad bos 3, belonging to the serological subgroup I, and Ad bos 4, Ad bos 5, Ad bos 7, Ad bos 8, belonging to the serological subgroup II, and those isolated from animal adenoviruses N18 and N3056. Cytomorphological method is supposed to be used not only for revealing bovine adenoviruses but also for determining preliminarily their subgroup belonging. PMID:3754069

  18. ANTIGEN DETECTION WITH MONOCLONAL ANTIBODIES FOR THE DIAGNOSIS OF ADENOVIRUS GASTROENTERITIS

    EPA Science Inventory

    The authors have developed a monoclonal antibody-based enzyme immunoassay (EIA) for direct detection of enteric adenoviruses in stool specimens from patients with gastroenteritis. Tests specific for each of the enteric adenoviruses, adenovirus type 40 (Ad40) and type 41 (Ad41) we...

  19. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Smythe, W R; Hwang, H C; Elshami, A A; Amin, K M; Eck, S L; Davidson, B L; Wilson, J M; Kaiser, L R; Albelda, S M

    1995-01-01

    OBJECTIVE: The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. BACKGROUND DATA: Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. METHODS: An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. RESULTS: Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. CONCLUSION: These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and

  20. Chemical Modification with High Molecular Weight Polyethylene Glycol Reduces Transduction of Hepatocytes and Increases Efficacy of Intravenously Delivered Oncolytic Adenovirus

    PubMed Central

    Doronin, Konstantin; Shashkova, Elena V.; May, Shannon M.; Hofherr, Sean E.

    2009-01-01

    Abstract Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites. PMID:19469693

  1. Modification of Ad5 Hexon Hypervariable Regions Circumvents Pre-Existing Ad5 Neutralizing Antibodies and Induces Protective Immune Responses

    PubMed Central

    Bruder, Joseph T.; Semenova, Elena; Chen, Ping; Limbach, Keith; Patterson, Noelle B.; Stefaniak, Maureen E.; Konovalova, Svetlana; Thomas, Charlie; Hamilton, Melissa; King, C. Richter; Richie, Thomas L.; Doolan, Denise L.

    2012-01-01

    The development of an effective malaria vaccine is a high global health priority. Vaccine vectors based on adenovirus type 5 are capable of generating robust and protective T cell and antibody responses in animal models and are currently being evaluated in clinical trials for HIV and malaria. They appear to be more effective in terms of inducing antigen-specific immune responses as compared with non-Ad5 serotype vectors. However, the high prevalence of neutralizing antibodies to Ad5 in the human population, particularly in the developing world, has the potential to limit the effectiveness of Ad5-based vaccines. We have generated novel Ad5-based vectors that precisely replace the hexon hypervariable regions with those derived from Ad43, a subgroup D serotype with low prevalence of neutralizing antibody in humans. We have demonstrated that these hexon-modified adenovectors are not neutralized efficiently by Ad5 neutralizing antibodies in vitro using sera from mice, rabbits and human volunteers. We have also generated hexon-modified adenovectors that express a rodent malaria parasite antigen, PyCSP, and demonstrated that they are as immunogenic as an unmodified vector. Furthermore, in contrast to the unmodified vector, the hexon-modified adenovectors induced robust T cell responses in mice with high levels of Ad5 neutralizing antibody. We also show that the hexon-modified vector can be combined with unmodified Ad5 vector in prime-boost regimens to induce protective responses in mice. Our data establish that these hexon-modified vectors are highly immunogenic even in the presence of pre-existing anti-adenovirus antibodies. These hexon-modified adenovectors may have advantages in sub-Saharan Africa where there is a high prevalence of Ad5 neutralizing antibody in the population. PMID:22496772

  2. Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector

    PubMed Central

    Bukh, Irene; Calcedo, Roberto; Roy, Soumitra; Carnathan, Diane G.; Grant, Rebecca; Qin, Qiuyue; Boyd, Surina; Ratcliffe, Sarah J.; Veeder, Christin L.; Bellamy, Scarlett L.; Betts, Michael R.

    2014-01-01

    ABSTRACT The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV

  3. Adenoviral vector-based strategies against infectious disease and cancer

    PubMed Central

    Zhang, Chao; Zhou, Dongming

    2016-01-01

    ABSTRACT Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vector-based strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. PMID:27105067

  4. Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine.

    PubMed

    Jones, Frank R; Gabitzsch, Elizabeth S; Xu, Younong; Balint, Joseph P; Borisevich, Viktoriya; Smith, Jennifer; Smith, Jeanon; Peng, Bi-Hung; Walker, Aida; Salazar, Magda; Paessler, Slobodan

    2011-09-16

    Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases. PMID:21821082

  5. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  6. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Canine Hepatitis and Canine Adenovirus...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  7. Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy.

    PubMed

    Choi, Joung-Woo; Park, Ji Won; Na, Youjin; Jung, Soo-Jung; Hwang, June Kyu; Choi, Dongho; Lee, Kyeong Geun; Yun, Chae-Ok

    2015-10-01

    Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy. PMID:26164117

  8. Impact of Adenovirus infection in host cell metabolism evaluated by (1)H-NMR spectroscopy.

    PubMed

    Silva, Ana Carina; P Teixeira, Ana; M Alves, Paula

    2016-08-10

    Adenovirus-based vectors are powerful vehicles for gene transfer applications in vaccination and gene therapy. Although highly exploited in the clinical setting, key aspects of the adenovirus biology are still not well understood, in particular the subversion of host cell metabolism during viral infection and replication. The aim of this work was to gain insights on the metabolism of two human cell lines (HEK293 and an amniocyte-derived cell line, 1G3) after infection with an adenovirus serotype 5 vector (AdV5). In order to profile metabolic alterations, we used (1)H-NMR spectroscopy, which allowed the quantification of 35 metabolites in cell culture supernatants with low sample preparation and in a relatively short time. Significant differences between both cell lines in non-infected cultures were identified, namely in glutamine and acetate metabolism, as well as by-product secretion. The main response to AdV5 infection was an increase in glucose consumption and lactate production rates. Moreover, cultures performed with or without glutamine supplementation confirmed the exhaustion of this amino acid as one of the main causes of lower AdV5 production at high cell densities (10- and 1.5-fold less specific yields in HEK293 and 1G3 cells, respectively), and highlighted different degrees of glutamine dependency of adenovirus replication in each cell line. The observed metabolic alterations associated with AdV5 infection and specificity of the host cell line can be useful for targeted bioprocess optimization. PMID:27215342

  9. Artificial envelopment of nonenveloped viruses: enhancing adenovirus tumor targeting in vivo.

    PubMed

    Singh, Ravi; Tian, Bowen; Kostarelos, Kostas

    2008-09-01

    Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad by systemic administration is limited due to rapid clearance from blood circulation, transfection of nontarget tissues, toxicity, and immunogenicity. To address these limitations, we developed an artificially enveloped Ad vector prepared by self-assembly of lipid bilayers around the Ad capsid. The physicochemical and structural features of the enveloped Ad vector can be altered according to the type of lipid used without the need for genetic modification or conjugation chemistry. Here we engineered 4 different types of artificially enveloped Ad using cationic, neutral, fusogenic, and PEGylated lipids to form the envelopes and obtained extended blood circulation times following i.v. administration and reduced vector immunogenicity. Moreover, the PEGylated lipid-enveloped Ad was capable of specifically delivering genes via the systemic circulation to solid tumors subcutaneously implanted in the absence of high levels of gene transfer to the liver and spleen. This provides the basis for the development of a novel vector platform for systemic delivery of Ad to disseminated targets. Furthermore, the artificial envelopment of Ad presented herein is an illustration of a general strategy to modulate the biological function of nonenveloped viruses and may have implications broader than gene therapy. PMID:18556649

  10. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  11. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker. PMID:26723876

  12. Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

    PubMed Central

    Scala, Stefania; Portella, Giuseppe; Fedele, Monica; Chiappetta, Gennaro; Fusco, Alfredo

    2000-01-01

    High mobility group I (HMGI) proteins are overexpressed in several human malignant tumors. We previously demonstrated that inhibition of HMGI synthesis prevents thyroid cell transformation. Here, we report that an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas) induced programmed cell death of two human thyroid anaplastic carcinoma cell lines (ARO and FB-1), but not normal thyroid cells. The Ad-Yas virus led to death of lung, colon, and breast carcinoma cells. A control adenovirus carrying the lacZ gene did not inhibit the growth of either normal or neoplastic cells. Ad-Yas treatment of tumors induced in athymic mice by ARO cells caused a drastic reduction in tumor size. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may be a useful treatment strategy in a variety of human malignant neoplasias, in which HMGI(Y) gene overexpression is a general event. PMID:10759549

  13. Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

    PubMed Central

    Lam, Eric

    2014-01-01

    ABSTRACT The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. IMPORTANCE This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions

  14. Dimension Six Correction to the Vector Sector of AdS/QCD Model

    SciTech Connect

    Hovhannes Grigoryan

    2007-09-06

    We study the effect of dimension six term F^3 on the predictions of the holographic model for the vector meson form factor. It is shown that the dimensionless parameter with which this term enters the action determines the corrections to the electric radius, magnetic and quadrupole moments of the rho meson. The results suggest that the addition of higher order terms may improve the holographic model.

  15. Short-fiber protein of ad40 confers enteric tropism and protection against acidic gastrointestinal conditions.

    PubMed

    Rodríguez, Ester; Romero, Carolina; Río, Adolfo; Miralles, Marta; Raventós, Aida; Planells, Laura; Burgueño, Joan F; Hamada, Hirofumi; Perales, Jose Carlos; Bosch, Assumpció; Gassull, Miguel Angel; Fernández, Ester; Chillon, Miguel

    2013-08-01

    The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter β-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases. PMID:23746215

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

    SciTech Connect

    Puntel, Mariana; Ghulam, Muhammad A.K.M.; Farrokhi, Catherine; VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley; Kroeger, Kurt M.; Salem, Alireza; Lacayo, Liliana; Pechnick, Robert N.; Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean; Palmer, Donna; Ng, Philip; and others

    2013-05-01

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

  17. Localization of neutralization epitopes on adenovirus fiber knob from species C.

    PubMed

    Lang, Shuai; Wang, Lizheng; Wang, Zixuan; Zhu, Rui; Yan, Jingyi; Wang, Baoming; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Zhou, Yan; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-04-01

    Although potential neutralization epitopes on the fiber knob of adenovirus (AdV) serotype 2 (Ad2) and Ad5 have been revealed, few studies have been carried out to identify neutralization epitopes on the knob from a broader panel of AdV serotypes. In this study, based on sequence and structural analysis of knobs from Ad1, Ad2, Ad5 and Ad6 (all from species C), several trimeric chimeric knob proteins were expressed in Escherichia coli to identify the locations of neutralization epitopes on the knobs by analysing their reactivity with mouse and rabbit polyclonal sera raised against AdVs and human sera with natural AdV infection. The dominant neutralization epitopes were located mainly in the N-terminal part of knobs from Ad1, Ad2 and Ad5, but they seemed to be located in the C-terminal part of the Ad6 knob, with some individual differences in rabbit and human populations. Our study adds to our understanding of humoral immune responses to AdVs and will facilitate the construction of more desirable capsid-modified recombinant Ad5 vectors. PMID:26801881

  18. Frontiers for the Early Diagnosis of AD by Means of MRI Brain Imaging and Support Vector Machines.

    PubMed

    Salvatore, Christian; Battista, Petronilla; Castiglioni, Isabella

    2016-01-01

    The emergence of Alzheimer's Disease (AD) as a consequence of increasing aging population makes urgent the availability of methods for the early and accurate diagnosis. Magnetic Resonance Imaging (MRI) could be used as in vivo, non invasive tool to identify sensitive and specific markers of very early AD progression. In recent years, multivariate pattern analysis (MVPA) and machine- learning algorithms have attracted strong interest within the neuroimaging community, as they allow automatic classification of imaging data with higher performance than univariate statistical analysis. An exhaustive search of PubMed, Web of Science and Medline records was performed in this work, in order to retrieve studies focused on the potential role of MRI in aiding the clinician in early diagnosis of AD by using Support Vector Machines (SVMs) as MVPA automated classification method. A total of 30 studies emerged, published from 2008 to date. This review aims to give a state-of-the-art overview about SVM for the early and differential diagnosis of AD-related pathologies by means of MRI data, starting from preliminary steps such as image pre-processing, feature extraction and feature selection, and ending with classification, validation strategies and extraction of MRI-related biomarkers. The main advantages and drawbacks of the different techniques were explored. Results obtained by the reviewed studies were reported in terms of classification performance and biomarker outcomes, in order to shed light on the parameters that accompany normal and pathological aging. Unresolved issues and possible future directions were finally pointed out. PMID:26567735

  19. Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than serotype 5: Implications for ovarian cancer therapy

    SciTech Connect

    Thoma, Clemens; Bachy, Veronique; Seaton, Patricia; Green, Nicola K.; Greaves, David R.; Klavinskis, Linda; Seymour, Leonard W.; Morrison, Joanne

    2013-12-15

    In a phase II/III clinical trial intraperitoneal (i.p.) administration of a group C adenovirus vector (Ad5) caused bowel adhesion formation, perforation and obstruction. However, we had found that i.p. group B, in contrast to group C adenoviruses, did not cause adhesions in nude BALB/c ovarian cancer models, prompting further investigation. Ex vivo, group B Ad11 caused lower inflammatory responses than Ad5 on BALB/c peritoneal macrophages. In vivo, i.p. Ad11 triggered short-term cytokine and cellular responses equal to Ad5 in both human CD46-positive and -negative mice. In contrast, in a long-term study of repeated i.p. administration, Ad11 caused no/mild, whereas Ad5 induced moderate/severe adhesions and substantial liver toxicity accompanied by elevated levels of IFNγ and VEGF and loss of i.p. macrophages, regardless of CD46 expression. It appears that, although i.p. Ad11 evokes immediate inflammation similar to Ad5, repeated administration of Ad11 is better tolerated and long-term fibrotic tissue remodelling is reduced. - Highlights: • i.p. Ad11 causes less long-term intraperitoneal inflammation than Ad5 in CD46-transgenic mice. • Ex vivo BALB/c peritoneal macrophages express less RANTES after Ad11 than Ad3 or Ad5 treatment. • In vivo, cytokine and cellular responses 6 h after i.p. Ad11 are equal to Ad5. • In contrast, after repeated i.p. application, Ad5, but not Ad11, causes severe i.p. toxicity. • The use of Ad11 instead of Ad5 might increase patient safety in future virotherapy of ovarian cancer.

  20. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

  1. [Inhibition of adenovirus reproduction in cell culture by specific antibodies].

    PubMed

    Povnytsia, O Iu; Nosach, L M; Zhovnovata, V L; Zahorodnia, S D; Vantsak, N P; Tokarchuk, L V; Polishchuk, O M; Diachenko, N S

    2009-01-01

    The capacity of specific antibodies to inhibit the reproduction of homo- and heterologous adenoviruses in Hela cell added to culture medium after virus adsorption was studied. The inhibiting effect of polyclonal antivirus and monospecific antihexone antibodies to homo- and heterologous adenoviruses was shown. The effect was more expressed when using antibodies to homologous antibodies. The intensity of inhibition depended on antibodies concentration in the medium and infecting dose of the virus. Essential reduction of the quantity of infected cells and a decrease of the titer of adenovirus synthesized in the presence of homo- and heterologous antibodies was shown but adenovirus reproduction was not inhibited completely. PMID:19663330

  2. RAD51 and BRCA2 enhance oncolytic adenovirus type 5 activity in ovarian cancer

    PubMed Central

    Tookman, Laura A.; Browne, Ashley K.; Connell, Claire M.; Bridge, Gemma; Ingemarsdotter, Carin K.; Dowson, Suzanne; Shibata, Atsushi; Lockley, Michelle; Martin, Sarah A.; McNeish, Iain A.

    2015-01-01

    Homologous Recombination (HR) function is critically important in High Grade Serous Ovarian Cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall anti-tumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2 mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. Implications Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function. PMID:26452665

  3. Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model.

    PubMed

    Kuryk, Lukasz; Haavisto, Elina; Garofalo, Mariangela; Capasso, Cristian; Hirvinen, Mari; Pesonen, Sari; Ranki, Tuuli; Vassilev, Lotta; Cerullo, Vincenzo

    2016-10-15

    Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma. PMID:27287512

  4. Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene

    PubMed Central

    Zhao, Chen; Wu, Ningning; Deng, Fang; Zhang, Hongmei; Wang, Ning; Zhang, Wenwen; Chen, Xian; Wen, Sheng; Zhang, Junhui; Yin, Liangjun; Liao, Zhan; Zhang, Zhonglin; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Wu, Di; Ye, Jixing; Deng, Youlin; Zhou, Guolin; Luu, Hue H.; Haydon, Rex C.; Si, Weike; He, Tong-Chuan

    2014-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells. PMID:24658746

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

    PubMed

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

    2016-05-01

    In a Phase 1 trial, we evaluated the safety of AERAS-402, an adenovirus 35-vectored TB vaccine candidate expressing 3 Mycobacterium tuberculosis (Mtb) immunodominant antigens, in subjects with and without latent Mtb infection. HIV-negative, BCG-vaccinated Kenyan adults without evidence of tuberculosis, 10 QuantiFERON(®)-TB Gold In-Tube test (QFT-G)(-) and 10 QFT-G(+), were randomized 4:1 to receive AERAS-402 or placebo as two doses, on Days 0 and 56, with follow up to Day 182. There were no deaths, serious adverse events or withdrawals. For 1 AERAS-402 QFT-G(-) and 1 AERAS-402 QFT-G(+) subject, there were 3 self-limiting severe AEs of injection site pain: 1 after the first vaccination and 1 after each vaccination, respectively. Two additional severe AEs considered vaccine-related were reported after the first vaccination in AERAS-402 QFT-G(+) subjects: elevated blood creatine phosphokinase and neutropenia, the latter slowly improving but remaining abnormal until study end. AERAS-402 was not detected in urine or throat cultures for any subject. In intracellular cytokine staining studies, curtailed by technical issues, we saw modest CD4+ and CD8+ T cell responses to Mtb Ag85A/b peptide pools among both QFT-G(-) and (+) subjects, with trends in the CD4+ T cells suggestive of boosting after the second vaccine dose, slightly more so in QFT-G(+) subjects. CD4+ and CD8+ responses to Mtb antigen TB10.4 were minimal. Increases in Adenovirus 35 neutralizing antibodies from screening to end of study, seen in 50% of AERAS-402 recipients, were mostly minimal. This small study confirms acceptable safety and tolerability profiles for AERAS-402, in line with other Phase 1 studies of AERAS-402, now to include QFT-G(+) subjects. PMID:27026148

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

    PubMed Central

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

    2014-01-01

    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 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. PMID:24473833

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

    PubMed

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

    2010-07-01

    Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines. PMID:20472025

  8. Adenovirus Type 2-Simian Virus 40 Hybrid Population: Evidence for a Hybrid Deoxyribonucleic Acid Molecule and the Absence of Adenovirus-Encapsidated Circular Simian Virus 40 Deoxyribonucleic Acid

    PubMed Central

    Crumpacker, Clyde S.; Levin, Myron J.; Wiese, William H.; Lewis, Andrew M.; Rowe, Wallace P.

    1970-01-01

    The deoxyribonucleic acid (DNA) from the adenovirus-encapsidated particles of the adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid population plaque variant (Ad2++ HEY), known to yield SV40 virus with high efficiency, was studied by equilibrium density centrifugation followed by ribonucleic acid-DNA hybridization employing virus-specific complementary ribonucleic acids synthesized in vitro. These techniques establish linkage between the Ad2 and SV40 components in the adenovirus-encapsidated particles of this population. The linkage is alkali-resistant and presumably covalent; thus, the Ad2 DNA and SV40 DNA are present in a hybrid molecule. Velocity centrifugation studies in alkaline sucrose gradients eliminated the possibility that supercoiled circular SV40 DNA is present in the adenovirus capsids. The DNA obtained from the adenovirus-encapsidated particles of the Ad2++ HEY population appears to consist of nonhybrid Ad2 DNA and Ad2-SV40 hybrid DNA molecules. PMID:4322081

  9. Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

    PubMed

    Wu, Qian; Liu, Wenjun; Xu, Bi; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2016-02-01

    A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples. PMID:26526147

  10. Mechanism of Ad5 Vaccine Immunity and Toxicity: Fiber Shaft Targeting of Dendritic Cells

    PubMed Central

    Kong, Wing-pui; Sheets, Rebecca L; Gomez, Phillip L; King, C. Richter; Nabel, Gary J

    2007-01-01

    Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs) was independent of the coxsackievirus and adenovirus receptor (CAR), its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines. PMID:17319743

  11. Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35▿ †

    PubMed Central

    Kälin, Stefan; Amstutz, Beat; Gastaldelli, Michele; Wolfrum, Nina; Boucke, Karin; Havenga, Menzo; DiGennaro, Fabienne; Liska, Nicole; Hemmi, Silvio; Greber, Urs F.

    2010-01-01

    Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, αν integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells. PMID:20237079

  12. CD46-Mediated Transduction of a Species D Adenovirus Vaccine Improves Mucosal Vaccine Efficacy

    PubMed Central

    Camacho, Zenaido T.; Turner, Mallory A.; Barry, Michael A.

    2014-01-01

    Abstract The high levels of preexisting immunity against Adenovirus type 5 (Ad5) have deemed Ad5 unusable for translation as a human vaccine vector. Low seroprevalent alternative viral vectors may be less impacted by preexisting immunity, but they may also have significantly different phenotypes from that of Ad5. In this study we compare species D Ads (26, 28, and 48) to the species C Ad5. In vitro transduction studies show striking differences between the species C and D viruses. Most notably, Ad26 transduced human dendritic cells much more effectively than Ad5. In vivo imaging studies showed strikingly different transgene expression profiles. The Ad5 virus was superior to the species D viruses in BALB/c mice when delivered intramuscularly. However, the inverse was true when the viruses were delivered mucosally via the intranasal epithelia. Intramuscular transduction was restored in mice that ubiquitously expressed human CD46, the primary receptor for species D viruses. We analyzed both species C and D Ads for their ability to induce prophylactic immunity against influenza in the CD46 transgenic mouse model. Surprisingly, the species D vaccines again failed to induce greater levels of protective immunity as compared with the species C Ad5 when delivered intramuscularly. However, the species D Ad vaccine vector, Ad48, induced significantly greater protection as compared with Ad5 when delivered mucosally via the intranasal route in CD46 transgenic mice. These data shed light on the complexities between the species and types of Ad. Our findings indicate that more research will be required to identify the mechanisms that play a key role in the induction of protective immunity induced by species D Ad vaccines. PMID:24635714

  13. CD46-mediated transduction of a species D adenovirus vaccine improves mucosal vaccine efficacy.

    PubMed

    Camacho, Zenaido T; Turner, Mallory A; Barry, Michael A; Weaver, Eric A

    2014-04-01

    The high levels of preexisting immunity against Adenovirus type 5 (Ad5) have deemed Ad5 unusable for translation as a human vaccine vector. Low seroprevalent alternative viral vectors may be less impacted by preexisting immunity, but they may also have significantly different phenotypes from that of Ad5. In this study we compare species D Ads (26, 28, and 48) to the species C Ad5. In vitro transduction studies show striking differences between the species C and D viruses. Most notably, Ad26 transduced human dendritic cells much more effectively than Ad5. In vivo imaging studies showed strikingly different transgene expression profiles. The Ad5 virus was superior to the species D viruses in BALB/c mice when delivered intramuscularly. However, the inverse was true when the viruses were delivered mucosally via the intranasal epithelia. Intramuscular transduction was restored in mice that ubiquitously expressed human CD46, the primary receptor for species D viruses. We analyzed both species C and D Ads for their ability to induce prophylactic immunity against influenza in the CD46 transgenic mouse model. Surprisingly, the species D vaccines again failed to induce greater levels of protective immunity as compared with the species C Ad5 when delivered intramuscularly. However, the species D Ad vaccine vector, Ad48, induced significantly greater protection as compared with Ad5 when delivered mucosally via the intranasal route in CD46 transgenic mice. These data shed light on the complexities between the species and types of Ad. Our findings indicate that more research will be required to identify the mechanisms that play a key role in the induction of protective immunity induced by species D Ad vaccines. PMID:24635714

  14. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

    PubMed Central

    Wickham, T J; Segal, D M; Roelvink, P W; Carrion, M E; Lizonova, A; Lee, G M; Kovesdi, I

    1996-01-01

    A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer. PMID:8794324

  15. Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

    PubMed

    Jung, Soo-Jung; Kasala, Dayananda; Choi, Joung-Woo; Lee, Soo-Hwan; Hwang, June Kyu; Kim, Sung Wan; Yun, Chae-Ok

    2015-01-12

    Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy. PMID:25400213

  16. Cryo-EM structures of two bovine adenovirus type 3 intermediates

    SciTech Connect

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

    2014-02-15

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

  17. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGESBeta

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; et al

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  18. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

  19. Protection against Mucosal SHIV Challenge by Peptide and Helper-Dependent Adenovirus Vaccines

    PubMed Central

    Weaver, Eric A.; Nehete, Pramod N.; Nehete, Bharti P.; Buchl, Stephanie J.; Palmer, Donna; Montefiori, David C.; Ng, Philip; Sastry, K. Jagannadha; Barry, Michael A.

    2009-01-01

    Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helper-dependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5-immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in the HD-Ad5 group. In this study, neutralizing antibody and T cell responses were compared between the groups before and after rectal challenge with CCR5-tropic SHIV-SF162P3. When serum samples were assayed for neutralizing antibodies, only weak activity was observed. T cell responses against env epitopes were higher in the serotype-switched group. When these animals were challenged rectally with SHIV-SF162P3, both the Ad5 and serotype-switch groups significantly reduced peak viral loads 2 to 10-fold 2 weeks after infection. Peak viral loads were significantly lower for the serotype-switched group as compared to the HD-Ad5-immunized group. Viral loads declined over 18 weeks after infection with some animals viremia reducing nearly 4 logs from the peak. These data demonstrate significant mucosal vaccine effects after immunization with only env antigens. These data also demonstrate HD-Ad vectors are a robust platform for vaccination. PMID:20107521

  20. Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through two interspecies recombination events

    PubMed Central

    Dehghan, Shoaleh; Seto, Jason; Liu, Elizabeth B.; Walsh, Michael P.; Dyer, David W.; Chodosh, James; Seto, Donald

    2013-01-01

    Computational analysis of human adenovirus type 4 (HAdV-E4), a pathogen that is the only HAdV member of species E, provides insights into its zoonotic origin and molecular adaptation. Its genome encodes a domain of the major capsid protein, hexon, from HAdV-B16 recombined into the genome chassis of a simian adenovirus. Genomes of two recent field strains provide a clue to its adaptation to the new host: recombination of a NF-I binding site motif, which is required for efficient viral replication, from another HAdV genome. This motif is absent in the chimpanzee adenoviruses and the HAdV-E4 prototype, but is conserved amongst other HAdVs. This is the first report of an interspecies recombination event for HAdVs, and the first documentation of a lateral partial gene transfer from a chimpanzee AdV. The potential for such recombination events are important when considering chimpanzee adenoviruses as candidate gene delivery vectors for human patients. PMID:23763770

  1. ENHANCEMENT OF ADENOVIRUS DELIVERY AFTER ULTRASOUND-STIMULATED THERAPY IN A CANCER MODEL

    PubMed Central

    Sorace, Anna G.; Warram, Jason M.; Mahoney, Marshall; Zinn, Kurt R.; Hoyt, Kenneth

    2014-01-01

    Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. In vitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for In vivo analysis to quantify Ad infection within the tumor. In vitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p = 0.80). In vivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1 × 106 plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p = 0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues. PMID:24063960

  2. Physical organization of subgroup B human adenovirus genomes.

    PubMed Central

    Tibbetts, C

    1977-01-01

    Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved. Images PMID:916027

  3. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO ENTERIC ADENOVIRUS TYPES 40 AND 41

    EPA Science Inventory

    The authors have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad4...

  4. Efficient lung orthotopic tumor-growth suppression of oncolytic adenovirus complexed with RGD-targeted bioreducible polymer.

    PubMed

    Kim, J; Nam, H Y; Choi, J W; Yun, C-O; Kim, S W

    2014-05-01

    Oncolytic adenoviruses (Ad) have been developed for the eradication of tumors. Although they hold much promise as a cancer therapy, they have a short blood circulation time and high liver toxicity. An effective strategy to overcome these problems has been complexing Ad with shielding materials. However, the therapeutic efficacy of the Ad complexes has also been an issue because passive accumulation does not allow for sufficient delivery of Ad to the cancer cells. To enhance the therapeutic efficacy of the polymer-coated Ads, the attachment of a targeting moiety to polymer-coated Ad vectors is inescapable. Our lab has previously reported the potential use of Arg-Gly-Asp (RGD)-targeted bioreducible polymers with a polyethylene glycol (PEG) linker for delivering oncolytic Ads. We have shown the enhanced in vitro transduction efficiency and increased cancer-killing effect with producing progeny oncolytic Ad particles. In addition, we have shown significant tumor-growth inhibition of the polymer-shielded Ad in an in vivo lung orthotopic tumor model. The shielding effect of the Ad surface with the polymers allowed evasion of host immune responses and reduction of liver toxicity. This data demonstrates that the RGD-conjugated bioreducible polymer for delivering the oncolytic Ad vectors could be utilized for cancer therapy via systemic administration. PMID:24598892

  5. Stimulation of innate immunity by in vivo cyclic di-GMP synthesis using adenovirus.

    PubMed

    Koestler, Benjamin J; Seregin, Sergey S; Rastall, David P W; Aldhamen, Yasser A; Godbehere, Sarah; Amalfitano, Andrea; Waters, Christopher M

    2014-11-01

    The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination. PMID:25230938

  6. Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization▿

    PubMed Central

    Cheng, Cheng; Gall, Jason G. D.; Nason, Martha; King, C. Richter; Koup, Richard A.; Roederer, Mario; McElrath, M. Juliana; Morgan, Cecilia A.; Churchyard, Gavin; Baden, Lindsey R.; Duerr, Ann C.; Keefer, Michael C.; Graham, Barney S.; Nabel, Gary J.

    2010-01-01

    A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors. PMID:19846512

  7. ChAd63-MVA–vectored Blood-stage Malaria Vaccines Targeting MSP1 and AMA1: Assessment of Efficacy Against Mosquito Bite Challenge in Humans

    PubMed Central

    Sheehy, Susanne H; Duncan, Christopher JA; Elias, Sean C; Choudhary, Prateek; Biswas, Sumi; Halstead, Fenella D; Collins, Katharine A; Edwards, Nick J; Douglas, Alexander D; Anagnostou, Nicholas A; Ewer, Katie J; Havelock, Tom; Mahungu, Tabitha; Bliss, Carly M; Miura, Kazutoyo; Poulton, Ian D; Lillie, Patrick J; Antrobus, Richard D; Berrie, Eleanor; Moyle, Sarah; Gantlett, Katherine; Colloca, Stefano; Cortese, Riccardo; Long, Carole A; Sinden, Robert E; Gilbert, Sarah C; Lawrie, Alison M; Doherty, Tom; Faust, Saul N; Nicosia, Alfredo; Hill, Adrian VS; Draper, Simon J

    2012-01-01

    The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1—results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets. PMID:23089736

  8. 78 FR 3906 - Prospective Grant of a Co-Exclusive License: Adenovirus-Based Controls and Calibrators for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-17

    ... Transfer of Genes to the Lung'', and US patent 6,136,594 (HHS Reference E-129-1991/1- US-03), issued... adenovirus vectors containing foreign DNA. Such vectors can be used for gene transfer, therapeutics,...

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

    PubMed

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

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle. PMID:12960972

  10. [Immunological evaluation of vector-expressed M2 and HA genes of H5N1 influenza virus in mice].

    PubMed

    Guo, Jianqiang; Yao, Lihong; Chen, Aijun; Xu, Yi; Liu, Xiaoyu; Shu, Yuelong; Zhang, Zhiqing

    2010-05-01

    We developed vectors expressing two antigen of H5N1 influenza virus. Based on the human H5N1 avian influenza virus strain A/Anhui/1/2005 isolated in China, we amplified the matrix protein 2 (M2) and Hemagglutinin (HA) genes by PCR and subcloned them into pStar vector to construct two genes co-expressing recombinant DNA vaccine pStar-M2/HA. After transfection of 293 cells with the plasmid, we confirmed with indirect immunofluorescence assay (IFA) that M2 and HA genes cloned on plasmid pStar co-expressed successfully. Using Ad-Easy adenovirus vector system, by homologous recombination in bacteria and packaging in 293 cells, we constructed two recombinant adenoviruses, namely Ad-M2 and Ad-HA. After infection of 293 cells with the recombinant adenoviruses, we confirmed with IFA that M2 and HA genes cloned into adenoviruses expressed successfully. We then combined the recombinant DNA vaccine and adenoviral vector vaccines in immunization of BALB/c mice with a prime-boost regime. On day 0 and day 28, we immunized the mice with DNA vaccine and on day 14 and day 42, with recombinant adenovirus vaccines. We took blood samples before each injection and 14 days after the final injection. On day 56, we collected splenocytes from the mice. ELISA and hemagglutination inhibition (HI) assay showed that the vaccines successfully induced specific IgG antibodies against HA protein in serum of the immunized mice. ELISPOT confirmed that the vaccines successfully induced the special cellular immune response to M2 and HA protein of H5N1 influenza virus. The study on combined immunization with M2 and HA genes provided basis for development of novel influenza vaccine. PMID:20684310

  11. Labeling of Adenovirus Particles with PARACEST Agents

    PubMed Central

    Vasalatiy, Olga; Gerard, Robert D; Zhao, Piyu; Sun, Xiankai; Sherry, A. Dean

    2009-01-01

    Recombinant adenovirus type 5 particles (AdCMVLuc) were labeled with two different bifunctional ligands capable of forming stable complexes with paramagnetic lanthanide ions. The number of covalently attached ligands varied between 630 and 1960 per adenovirus particle depending upon the chemical reactivity of the bifunctional ligand (NHS ester versus isothiocyanide), the amount of excess ligand added, and the reaction time. The bioactivity of each labeled adenovirus derivative, as measured by the ability of the virus to infect cells and express luciferase, was shown to be highly dependent upon the number of covalently attached ligands. This indicates that certain amino groups, likely on the surface of the adenovirus fiber protein where cell binding is known to occur, are critical for viral attachment and infection. Addition of 177Lu3+ to chemically modified versus control viruses demonstrated a significant amount of nonspecific binding of 177Lu3+ to the virus particles that could not be sequestered by addition of excess DTPA. Thus, it became necessary to implement a prelabeling strategy for conjugation of preformed lanthanide ligand chelates to adenovirus particles. Using preformed Tm3+-L2, a large number of chelates having chemical exchange saturation transfer (CEST) properties were attached to the surface residues of AdCMVLuc without nonspecific binding of metal ions elsewhere on the virus particle. The potential of such conjugates to act as PARACEST imaging agents was tested using an on-resonance WALTZ sequence for CEST activation. A 12% decrease in bulk water signal intensity was observed relative to controls. This demonstrates that viral particles labeled with PARACEST-type imaging agents can potentially serve as targeted agents for molecular imaging. PMID:18254605

  12. Different Patterns of Expansion, Contraction and Memory Differentiation of HIV-1 Gag-Specific CD8 T Cells Elicited by Adenovirus Type 5 and Modified Vaccinia Ankara Vaccines

    PubMed Central

    Pillai, Vinod Kumar Bhaskara; Kannanganat, Sunil; Penaloza-MacMaster, Pablo; Chennareddi, Lakshmi; Robinson, Harriet L.; Blackwell, Jerry; Amara, Rama Rao

    2011-01-01

    The magnitude and functional quality of antiviral CD8 T cell responses are critical for the efficacy of T cell based vaccines. Here, we investigate the influence of two popular viral vectors, adenovirus type 5 (Ad5) and modified vaccinia Ankara (MVA), on expansion, contraction and memory differentiation of HIV-1 Gag insert-specific CD8 T cell responses following immunization and show different patterns for the two recombinant viral vectors. The Ad5 vector primed 6-fold higher levels of insert-specific CD8 effector T cells than the MVA vector. The Ad5-primed effector cells also underwent less contraction (< 2-fold) than the MVA-primed cells (>5-fold). The Ad5-primed memory cells were predominantly CD62L negative (effector memory) whereas the MVA-primed memory cells were predominantly CD62L positive (central memory). Consistent with their memory phenotype, MVA-primed CD8 T cells underwent higher fold expansion than Ad5-primed CD8 T cells following a homologous or heterologous boost. Impressively, the Ad5 boost changed the quality of MVA-primed memory response such that they undergo less contraction with effector memory phenotype. However, the MVA boost did not influence the contraction and memory phenotype of Ad5-primed response. In conclusion, our results demonstrate that vaccine vector strongly influences the expansion, contraction and the functional quality of insert-specific CD8 T cell responses and have implications for vaccine development against infectious diseases. PMID:21651938

  13. NOD2 Signaling Contributes to the Innate Immune Response Against Helper-Dependent Adenovirus Vectors Independently of MyD88 In Vivo

    PubMed Central

    Suzuki, Masataka; Cela, Racel; Bertin, Terry K.; Sule, Gautam; Cerullo, Vincenzo; Rodgers, John R.

    2011-01-01

    Abstract We previously demonstrated that Toll-like receptor/myeloid differentiation primary response gene 88 (MyD88) signaling is required for maximal innate and acquired [T helper cell type 1 (Th1)] immune responses following systemic administration of helper-dependent adenoviral vectors (HDAds). However, MyD88-deficient mice injected with HDAdLacZ exhibited only partial reduction of innate immune cytokine expression compared with wild-type mice, suggesting MyD88-independent pathways also respond to HDAds. We now show that NOD2, a nucleotide-binding and oligomerization domain (NOD)–like receptor known to detect muramyl dipeptides in bacterial peptidoglycans, also contributes to innate responses to HDAds, but not to humoral or Th1 immune responses. We established NOD2/MyD88 double-deficient mice that, when challenged with HDAds, showed a significant reduction of the innate response compared with mice deficient for either gene singly, suggesting that NOD2 signaling contributes to the innate response independently of MyD88 signaling following systemic administration of HDAds. In addition, NOD2-deficient mice exhibited significantly higher transgene expression than did wild-type mice at an early time point (before development of an acquired response), but not at a later time point (after development of an acquired response). These results indicate that the intracellular sensor NOD2 is required for innate responses to HDAds and can limit transgene expression during early phases of infection. PMID:21561248

  14. Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract.

    PubMed

    Terao, R; Honda, K; Hatano, E; Uehara, T; Yamamoto, M; Yamaoka, Y

    1998-09-01

    Proliferative cholangitis (PC) associated with hepatolithiasis develops the stricture of main bile ducts, and is the main cause of residual and/or recurrent stones after repeated treatments for hepatolithiasis. The aim of this study was to inhibit PC using the cytostatic gene therapy with direct adenovirus-mediated retinoblastoma (Rb) gene transfer to the biliary tract. PC was induced by introducing a fine nylon thread into the bile duct in a rat model. The adenovirus vector encoding a nonphosphorylatable, constitutively active form of retinoblastoma gene product (AdRb) was administered directly into the biliary tract. The adenovirus vector encoding beta-galactosidase (AdlacZ) was also given as a control. The bile duct wall thickness and 5'-bromodeoxyuridine (BrdU) labeling index were compared among uninfected, AdlacZ-infected, and AdRb-infected PC rats. The Rb expression in the bile duct was detected using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical study. AdRb-infected bile ducts showed inhibition of the epithelial and fibrous tissue proliferation and the peribiliary gland hyperplasia, resulting in a significant reduction of wall thickness compared with uninfected and AdlacZ-infected ones. The BrdU labeling index was 4.87% +/- 3.06% in the AdRb-infected bile ducts, while those of uninfected and AdlacZ-infected ones were 15.48% +/- 4.61% and 11.72% +/- 1.23%, respectively (P < .05). In conclusion, our cytostatic gene therapy approach using direct Rb gene transfer into the biliary tract suppressed PC in a rat model and may offer an effective therapeutic option for reducing recurrences following treatments against hepatolithiasis. PMID:9731547

  15. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection.

    PubMed

    Wechman, Stephen L; Rao, Xiao-Mei; Cheng, Pei-Hsin; Gomez-Gutierrez, Jorge G; McMasters, Kelly M; Zhou, H Sam

    2016-01-01

    Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties. PMID:27314377

  16. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; Cheng, Pei-Hsin; Gomez-Gutierrez, Jorge G.; McMasters, Kelly M.; Zhou, H. Sam

    2016-01-01

    Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties. PMID:27314377

  17. Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV

    PubMed Central

    Nehete, Bharti P.; Yang, Guojun; Buchl, Stephanie J.; Hanley, Patrick W.; Palmer, Donna; Montefiori, David C.; Ferrari, Guido; Ng, Philip; Sastry, K. Jagannadha; Barry, Michael A.

    2013-01-01

    Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination. PMID:23844034

  18. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2004-05-18

    Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

  19. Mucosal immunization with recombinant adenovirus encoding soluble globular head of hemagglutinin protects mice against lethal influenza virus infection.

    PubMed

    Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H; Song, Man Ki; Chang, Jun

    2013-12-01

    Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection against the virus infection. Combination of recombinant adenovirus (rAd) vector-based vaccine and mucosal administration is a promising regimen for safe and effective vaccination against influenza. In this study, we constructed rAd encoding the globular head region of HA from A/Puerto Rico/8/34 virus as vaccine candidate. The rAd vaccine was engineered to express high level of the protein in secreted form. Intranasal or sublingual immunization of mice with the rAd-based vaccine candidates induced significant levels of sustained HA-specific mucosal IgA and IgG. When challenged with lethal dose of homologous virus, the vaccinated mice were completely protected from the infection. The results demonstrate that intranasal or sublingual vaccination with HA-encoding rAd elicits protective immunity against infection with homologous influenza virus. This finding underlines the potential of our recombinant adenovirus-based influenza vaccine candidate for both efficacy and rapid production. PMID:24385946

  20. Conserved Sequences at the Origin of Adenovirus DNA Replication

    PubMed Central

    Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

    1982-01-01

    The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

  1. Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

    PubMed Central

    Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

    2014-01-01

    Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

  2. Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle.

    PubMed

    Dulal, Pawan; Wright, Daniel; Ashfield, Rebecca; Hill, Adrian V S; Charleston, Bryan; Warimwe, George M

    2016-04-29

    Development of safe and efficacious vaccines whose potency is unaffected by long-term storage at ambient temperature would obviate major vaccine deployment hurdles and limit wastage associated with breaks in the vaccine cold chain. Here, we evaluated the immunogenicity of a novel chimpanzee adenovirus vectored Rift Valley Fever vaccine (ChAdOx1-GnGc) in cattle, following its thermostabilisation by slow desiccation on glass fiber membranes in the non-reducing sugars trehalose and sucrose. Thermostabilised ChAdOx1-GnGc vaccine stored for 6 months at 25, 37 or 45 ° C elicited comparable Rift Valley Fever virus neutralising antibody titres to those elicited by the 'cold chain' vaccine (stored at -80 ° C throughout) at the same dose, and these were within the range associated with protection against Rift Valley Fever in cattle. The results support the use of sugar-membrane thermostabilised vaccines in target livestock species. PMID:27020712

  3. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  4. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  5. One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

    PubMed

    Li, Li-Li; Wang, He-Rong; Zhou, Zhi-Yi; Luo, Jing; Xiao, Xiang-Qian; Wang, Xiao-Li; Li, Jin-Tao; Zhou, Yu-Bai; Zeng, Yi

    2016-04-01

    Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections. PMID:26821205

  6. In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

    PubMed

    Alyaqoub, Fadel S; Aldhamen, Yasser A; Koestler, Benjamin J; Bruger, Eric L; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Waters, Christopher M; Amalfitano, Andrea

    2016-02-15

    There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-β and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers. PMID:26792800

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene (AdTW68.H5ck). Commercial layer-type chicken groups were singly vaccinated ocularly at 5 days of age, or singly v...

  8. Structure of human adenovirus

    SciTech Connect

    Nemerow, Glen R.; Stewart, Phoebe L.; Reddy, Vijay S.

    2012-07-11

    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 {angstrom} resolution. The virus structure revealed the location, folds, and interactions of major and minor (cement proteins) on the inner and outer capsid surface. This new structural information sheds further light on the process of adenovirus capsid assembly and virus-host cell interactions.

  9. Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge.

    PubMed

    Hashem, Anwar; Jaentschke, Bozena; Gravel, Caroline; Tocchi, Monika; Doyle, Tracey; Rosu-Myles, Michael; He, Runtao; Li, Xuguang

    2012-04-01

    Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before. PMID:22370512

  10. Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung.

    PubMed

    Kaplan, J M; Armentano, D; Sparer, T E; Wynn, S G; Peterson, P A; Wadsworth, S C; Couture, K K; Pennington, S E; St George, J A; Gooding, L R; Smith, A E

    1997-01-01

    One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMV beta Gal vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the beta-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing beta-Gal from the E4+ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response. PMID:8989994

  11. Actin-resistant DNAse I Expression From Oncolytic Adenovirus Enadenotucirev Enhances Its Intratumoral Spread and Reduces Tumor Growth.

    PubMed

    Tedcastle, Alison; Illingworth, Sam; Brown, Alice; Seymour, Leonard W; Fisher, Kerry D

    2016-04-01

    Spread of oncolytic viruses through tumor tissue is essential to effective virotherapy. Interstitial matrix is thought to be a significant barrier to virus particle convection between "islands" of tumor cells. One way to address this is to encode matrix-degrading enzymes within oncolytic viruses, for secretion from infected cells. To test the hypothesis that extracellular DNA provides an important barrier, we assessed the ability of DNase to promote virus spread. Nonreplicating Ad5 vectors expressing actin-resistant DNase (aDNAse I), proteinase K (PK), hyaluronidase (rhPH20), and chondroitinase ABC (CABC) were injected into established DLD human colorectal adenocarcinoma xenografts, transcomplemented with a replicating Ad5 virus. Each enzyme improved oncolysis by the replicating adenovirus, with no evidence of tumor cells being shed into the bloodstream. aDNAse I and rhPH20 hyaluronidase were then cloned into conditionally-replicating group B adenovirus, Enadenotucirev (EnAd). EnAd encoding each enzyme showed significantly better antitumor efficacy than the parental virus, with the aDNAse I-expressing virus showing improved spread. Both DNase and hyaluronidase activity was still measurable 32 days postinfection. This is the first time that extracellular DNA has been implicated as a barrier for interstitial virus spread, and suggests that oncolytic viruses expressing aDNAse I may be promising candidates for clinical translation. PMID:26708004

  12. Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

    PubMed

    Zhang, Nian-Hua; Peng, Rui-Qing; Ding, Ya; Zhang, Xiao-Shi

    2016-08-01

    The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity. PMID:27373420

  13. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    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. PMID:25674795

  14. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle. PMID:25498212

  15. Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

    SciTech Connect

    Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam; Abu-Asab, Mones S.; Wildner, Oliver; Miles, Brian K.; Yim, Kevin C.; Ramanan, Vijay; Prince, Gregory A.; Morris, John C.

    2007-12-05

    Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

  16. The Evaluation of Polyhexamethylene Biguanide (PHMB) as a Disinfectant for Adenovirus

    PubMed Central

    Romanowski, Eric G.; Yates, Kathleen A.; O’Connor, Katherine E.; Mah, Francis S.; Shanks, Robert M. Q.; Kowalski, Regis P.

    2013-01-01

    Purpose Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs. The current study determined whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs. Methods The direct disinfecting activity of PHMB was determined in triplicate assays by incubating nine human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with 50 and 0 PPM (µg/ml) of PHMB for 24 hours at room temperature, to simulate swimming pool temperatures, or 40°C, to simulate hot tub temperatures. Plaque assays determined adenovirus titers after incubation. Titers were Log10 converted and mean ± standard deviation Log10 reductions from controls were calculated. Virucidal (greater than 99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested. Results At room temperature, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for all adenovirus types tested. At 40°C, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for two adenovirus types and greater than 1 Log10, but less than 3 Log10, for seven of nine adenovirus types. Conclusions 50 PPM of PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Clinical Relevance Recreational water maintained and sanitized with PHMB has the potential to serve as a vector for the transmission of ocular adenovirus infections. PMID:23450376

  17. Immunization with adenovirus LIGHT-engineered dendritic cells induces potent T cell responses and therapeutic immunity in HBV transgenic mice.

    PubMed

    Jiang, Wenzheng; Chen, Ran; Kong, Xiaobo; Long, Fengying; Shi, Yaru

    2014-07-31

    LIGHT, a TNF superfamily member (TNFSF14), is a type II transmembrane protein expressed on activated T cells and immature dendritic cells (DCs). However, the expression of LIGHT on mature DCs is down-regulated. Recent studies demonstrated that LIGHT provides potent costimulatory activity for T cells, enhancing proliferation and the production of Th1 cytokines independently of the B7-CD28 pathway. Here, we evaluated the effectiveness of peptide-pulsed DC-mediated antiviral immunity in HBV transgenic mice and the immunoadjuvant effect of LIGHT. The bone marrow-derived DCs were modified in vitro with an adenovirus (Ad) vector expressing mouse LIGHT (Ad-LIGHT), the expression of costimulatory molecules was up-regulated and the secretion of cytokines IL-12 and IFN-γ increased. LIGHT-modified DCs enhanced allostimulation for T cells in mixed lymphocyte reaction (MLR). HBV peptide-pulsed DCs elicited HBV specific CD8+ T cell response and reduced the level of HBsAg and HBV DNA in sera of HBV transgenic mice. Importantly, LIGHT-modified DCs could induce stronger antiviral immunity. These results support the concept that genetic modification of DCs with a recombinant LIGHT adenovirus vector may be a useful strategy for antiviral immunotherapy. PMID:24951859

  18. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    SciTech Connect

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

    2001-08-01

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

  19. Characterization of a novel adenovirus isolated from a skunk.

    PubMed

    Kozak, Robert A; Ackford, James G; Slaine, Patrick; Li, Aimin; Carman, Susy; Campbell, Doug; Welch, M Katherine; Kropinski, Andrew M; Nagy, Éva

    2015-11-01

    Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus. PMID:26189043

  20. Crystal Structure of Enteric Adenovirus Serotype 41 Short Fiber Head

    PubMed Central

    Seiradake, Elena; Cusack, Stephen

    2005-01-01

    Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding. PMID:16254343

  1. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

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

  2. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  3. PEGylated Adenoviruses: From Mice to Monkeys

    PubMed Central

    Wonganan, Piyanuch; Croyle, Maria A.

    2010-01-01

    Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models. PMID:21994645

  4. Dendritic cells serve as a “Trojan horse” for oncolytic adenovirus delivery in the treatment of mouse prostate cancer

    PubMed Central

    Li, Zhao-lun; Liang, Xuan; Li, He-cheng; Wang, Zi-ming; Chong, Tie

    2016-01-01

    Aim: Adenovirus-mediated gene therapy is a novel therapeutic approach for the treatment of cancer, in which replication of the virus itself is the anticancer method. However, the success of this novel therapy is limited due to inefficient delivery of the virus to the target sites. In this study, we used dendritic cells (DCs) as carriers for conditionally replicating adenoviruses (CRAds) in targeting prostate carcinoma (PCa). Methods: Four types of CRAds, including Ad-PC (without PCa-specific promoter and a recombinant human tumor necrosis factor, rmhTNF, sequence), Ad-PC-rmhTNF (without PCa-specific promoter), Ad-PPC-NCS (without an rmhTNF sequence) and Ad-PPC-rmhTNF, were constructed. The androgen-insensitive mouse PCa RM-1 cells were co-cultured with CRAd-loading DCs, and the viability of RM-1 cells was examined using MTT assay. The in vivo effects of CRAd-loading DCs on PCa were evaluated in RM-1 xenograft mouse model. Results: Two PCa-specific CRAds (Ad-PPC-NCS, Ad-PPC-rmhTNF) exhibited more potent suppression on the viability of RM-1 cells in vitro than the PCa-non-specific CRAds (Ad-PC, Ad-PC-rmhTNF). In PCa-bearing mice, intravenous injection of the PCa-specific CRAd-loading DCs significantly inhibited the growth of xenografted tumors, extended the survival time, and induced T-cell activation. Additionally, the rmhTNF-containing CRAds exhibited greater tumor killing ability than CRAds without rmhTNF. Conclusion: DCs may be an effective vector for the delivery of CRAds in the treatment of PCa. PMID:27345628

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

    PubMed Central

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

    2014-01-01

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

  6. The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry

    PubMed Central

    Loustalot, Fabien

    2015-01-01

    ABSTRACT The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FKCAV), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FKCAV entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family. PMID:26136571

  7. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

    PubMed

    Wang, Dan; Mou, Haiwei; Li, Shaoyong; Li, Yingxiang; Hough, Soren; Tran, Karen; Li, Jia; Yin, Hao; Anderson, Daniel G; Sontheimer, Erik J; Weng, Zhiping; Gao, Guangping; Xue, Wen

    2015-07-01

    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9. PMID:26086867

  8. The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

    PubMed Central

    Wodrich, Harald; Billet, Olivier; Perreau, Matthieu; Hippert, Claire; Mennechet, Franck; Schoehn, Guy; Lortat-Jacob, Hugues; Dreja, Hanna; Ibanes, Sandy; Kalatzis, Vasiliki; Wang, Jennifer P.; Finberg, Robert W.; Cusack, Stephen; Kremer, Eric J.

    2009-01-01

    Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models. PMID:19119424

  9. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy.

    PubMed

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-07-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer. PMID:27229159

  10. [Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

    PubMed

    Ma, Jing; Zhang, Xiao-Guang; Chen, Hong; Li, Kui-Biao; Zhang, Xiao-Mei; Zhang, Ke; Yang, Liang; Xu, Hong; Shu, Yue-Long; Tan, Wen-Jie; Zeng, Yi

    2009-09-01

    The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation. PMID:19954107

  11. Avian influenza vaccination in chickens and pigs with replication-competent adenovirus-free human recombinant adenovirus 5.

    PubMed

    Toro, Haroldo; van Ginkel, Frederik W; Tang, De-Chu C; Schemera, Bettina; Rodning, Soren; Newton, Joseph

    2010-03-01

    Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular vaccination with replication-competent adenovirus (RCA)-free human recombinant adenovirus serotype 5 (Ad5) encoding AI virus H5 (AdTW68.H5) or H7 (AdCN94.H7) hemagglutinins. We evaluated bivalent in ovo vaccination with AdTW68.H5 and AdCN94.H7 and determined that vaccinated chickens developed robust hemagglutination inhibition (HI) antibody levels to both H5 and H7 AI strains. Additionally, we evaluated immune responses of 1-day-old chickens vaccinated via spray with AdCN94.H7. These birds showed increased immunoglobulin A responses in lachrymal fluids and increased interleukin-6 expression in Harderian gland-derived lymphocytes. However, specific HI antibodies were not detected in the sera of these birds. Because pigs might play a role as a "mixing vessel" for the generation of pandemic influenza viruses we explored the use of RCA-free adenovirus technology to immunize pigs against AI virus. Weanling piglets vaccinated intramuscularly with a single dose of RCA-free AdTW68.H5 developed strong systemic antibody responses 3 wk postvaccination. Intranasal application of AdTW68.H5 in piglets resulted in reduced vaccine coverage, i.e., 33% of pigs (2/6) developed an antibody response, but serum antibody levels in those successfully immunized animals were similar to intramuscularly vaccinated animals. PMID:20521636

  12. Nucleic acid hybridization for detection of cell culture-amplified adenovirus.

    PubMed Central

    Huang, C; Deibel, R

    1988-01-01

    A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Seven cloned probes, as well as total adenovirus type 2 (Ad2) and Ad16 genomic DNA, were tested by a nucleic acid hybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. Adenovirus DNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, total Ad2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 reference serotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-H required less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2) and pAd16-BD (containing the BamHI D fragment from Ad16) hybridized only with reference serotypes from the homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected 100% of all isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-F were 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAd16-BD were 100% (subgroup B) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included in this study for comparison. Images PMID:3230138

  13. The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

    2014-08-01

    Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases. PMID:24955893

  14. On using multiple routing metrics with destination sequenced distance vector protocol for MultiHop wireless ad hoc networks

    NASA Astrophysics Data System (ADS)

    Mehic, M.; Fazio, P.; Voznak, M.; Partila, P.; Komosny, D.; Tovarek, J.; Chmelikova, Z.

    2016-05-01

    A mobile ad hoc network is a collection of mobile nodes which communicate without a fixed backbone or centralized infrastructure. Due to the frequent mobility of nodes, routes connecting two distant nodes may change. Therefore, it is not possible to establish a priori fixed paths for message delivery through the network. Because of its importance, routing is the most studied problem in mobile ad hoc networks. In addition, if the Quality of Service (QoS) is demanded, one must guarantee the QoS not only over a single hop but over an entire wireless multi-hop path which may not be a trivial task. In turns, this requires the propagation of QoS information within the network. The key to the support of QoS reporting is QoS routing, which provides path QoS information at each source. To support QoS for real-time traffic one needs to know not only minimum delay on the path to the destination but also the bandwidth available on it. Therefore, throughput, end-to-end delay, and routing overhead are traditional performance metrics used to evaluate the performance of routing protocol. To obtain additional information about the link, most of quality-link metrics are based on calculation of the lost probabilities of links by broadcasting probe packets. In this paper, we address the problem of including multiple routing metrics in existing routing packets that are broadcasted through the network. We evaluate the efficiency of such approach with modified version of DSDV routing protocols in ns-3 simulator.

  15. Human adenovirus-host cell interactions: comparative study with members of subgroups B and C.

    PubMed Central

    Defer, C; Belin, M T; Caillet-Boudin, M L; Boulanger, P

    1990-01-01

    Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus. Images PMID:2196380

  16. Retinal transduction profiles by high-capacity viral vectors

    PubMed Central

    Puppo, Agostina; Cesi, Giulia; Marrocco, Elena; Piccolo, Pasquale; Jacca, Sarah; Shayakhmetov, Dmitry M.; Parks, Robin J.; Davidson, Beverly L.; Colloca, Stefano; Brunetti-Pierri, Nicola; Ng, Philip; Donofrio, Gaetano; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5kb) genes. Viral vectors derived from Adenovirus (Ad), Lentivirus (LV) and Herpesvirus (HV) can package large DNA sequences but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes, and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium (RPE). We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8. PMID:24989814

  17. Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    PubMed Central

    Liu, Hui; Li, Lu; Meng, Haitao; Qian, Qijun

    2013-01-01

    An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis. PMID:23765161

  18. Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

    PubMed

    Hasegawa, K; Tagawa, M; Takagi, K; Tsukamoto, H; Tomioka, Y; Suzuki, T; Nishioka, Y; Ohrui, T; Numasaki, M

    2016-08-01

    Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy. PMID:27561689

  19. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  20. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  1. Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma

    PubMed Central

    2012-01-01

    Background Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model. Methods The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts. Results The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed. Conclusions These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma. PMID:23272637

  2. Longitudinal Requirement for CD4+ T Cell Help for Adenovirus Vector–Elicited CD8+ T Cell Responses

    PubMed Central

    Provine, Nicholas M.; Larocca, Rafael A.; Penaloza-MacMaster, Pablo; Borducchi, Erica N.; McNally, Anna; Parenteau, Lily R.; Kaufman, David R.

    2014-01-01

    Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8+ T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8+ T cell responses by Ad vector immunization is longitudinally dependent on CD4+ T cell help for a prolonged period. Depletion of CD4+ T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8+ T cells, decreased T-bet and eomesodermin expression, impaired KLRG1+ effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8+ T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4+ T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8+ T cells. These data demonstrate a prolonged temporal requirement for CD4+ T cell help for vaccine-elicited CD8+ T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8+ T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4+ T cell immune deficiencies. PMID:24778441

  3. Construction and radiolabeling of adenovirus variants that incorporate human metallothionein into protein IX for analysis of biodistribution.

    PubMed

    Liu, Lei; Rogers, Buck E; Aladyshkina, Natalia; Cheng, Bing; Lokitz, Stephen J; Curiel, David T; Mathis, J Michael

    2014-01-01

    Using adenovirus (Ad)-based vectors is a promising strategy for novel cancer treatments; however, current tracking approaches in vivo are limited. The C-terminus of the Ad minor capsid protein IX (pIX) can incorporate heterologous reporters to monitor biodistribution. We incorporated metallothionein (MT), a low-molecular-weight metal-binding protein, as a fusion to pIX. We previously demonstrated 99mTc binding in vitro to a pIX-MT fusion on the Ad capsid. We investigated different fusions of MT within pIX to optimize functional display. We identified a dimeric MT construct fused to pIX that showed significantly increased radiolabeling capacity. After Ad radiolabeling, we characterized metal binding in vitro. We explored biodistribution in vivo in control mice, mice pretreated with warfarin, mice preimmunized with wild-type Ad, and mice that received both warfarin pretreatment and Ad preimmunization. Localization of activity to liver and bladder was seen, with activity detected in spleen, intestine, and kidneys. Afterwards, the mice were euthanized and selected organs were dissected for further analysis. Similar to the imaging results, most of the radioactivity was found in the liver, spleen, kidneys, and bladder, with significant differences between the groups observed in the liver. These results demonstrate this platform application for following Ad dissemination in vivo. PMID:25060486

  4. In vivo model of adeno-associated virus vector persistence and rescue.

    PubMed Central

    Afione, S A; Conrad, C K; Kearns, W G; Chunduru, S; Adams, R; Reynolds, T C; Guggino, W B; Cutting, G R; Carter, B J; Flotte, T R

    1996-01-01

    Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue. PMID:8627804

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

    PubMed

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

    2016-07-01

    Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. PMID:27170642

  6. Experimental virotherapy of chemoresistant pancreatic carcinoma using infectivity-enhanced fiber-mosaic oncolytic adenovirus

    PubMed Central

    Kaliberov, Sergey A.; Kaliberova, Lyudmila N.; Buchsbaum, Donald J.; Curiel, David T.

    2014-01-01

    Pancreatic cancer is a significant clinical problem and novel therapeutic approaches are desperately needed. Recent advances in conditionally replicative adenovirus-based (CRAd) oncolytic virus design allow the application of CRAd vectors as a therapeutic strategy to efficiently target and eradicate chemoresistant pancreatic cancer cells thereby improving the efficacy of pancreatic cancer treatment. The goal of this study was to construct and validate the efficacy of an infectivity-enhanced, liver-untargeted, tumor-specific CRAd vector. A panel of CRAds has been derived which embody the C-X-C chemokine receptor type 4 promoter for conditional replication, two fiber complex mosaicism for targeting expansion, and hexon hypervariable region 7 (HVR7) modification for liver untargeting. We evaluated CRAds for cancer virotherapy using a human pancreatic tumor xenograft model. Employment of the fiber mosaic approach improved CRAd replication in pancreatic tumor xenografts. Substitution of the HVR7 of the Ad5 hexon for Ad serotype 3 hexon resulted in decreased liver tropism of systemically administrated CRAd. Obtained data demonstrated that employment of complex mosaicism increased efficacy of the combination of oncolytic virotherapy with chemotherapy in a human pancreatic tumor xenograft model. PMID:24903014

  7. The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

    PubMed Central

    Gabitzsch, Elizabeth S.; Tsang, Kwong Yok; Palena, Claudia; David, Justin M.; Fantini, Massimo; Kwilas, Anna; Rice, Adrian E.; Latchman, Yvette; Hodge, James W.; Gulley, James L.; Madan, Ravi A.; Heery, Christopher R.; Balint, Joseph P.

    2015-01-01

    Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies. PMID:26374823

  8. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

    PubMed

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

    2016-01-01

    Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. PMID:26499044

  9. [Producing Ad-IFN gamma by suspension culture of HEK293 cells in a disposable bioreactor].

    PubMed

    Wu, Quande; Huang, Wenlin

    2014-11-01

    Adenovirus vectors are promising delivery systems for gene therapy. We established a new process for clinic trial of recombinant adenovirus vectors using a novel disposable bioreactor. The suspension HEK293 cells were inoculated into a 5 L disposable bioreactor with parameters control of pH, DO, agitation and temperature. After 6 days of a fed-batch culture, the final cell density reached 2.0 x 10(6) cells/mL. The culture was infected with Ad-IFNγ at an MOI of 30. The harvest was performed at approximately 48 h post-infection and crude viral lysate was obtained after 3 freeze/thaw cycles and centrifugation. The maximum titers of crude viral lysate was 1.49 x 10(13) Infectious units (IFU) and the bulk product specific was 3,800 IFU/cell. Purified Ad-IFNγ by anion-exchange chromatography and the final recovery of infectious unit reached 35.9%. The result demonstrates that an efficient and stable process of producing Ad-IFNγ using a disposable fed-batch bioreactor is established. PMID:25985530

  10. Adenovirus-mediated artificial MicroRNAs targeting matrix or nucleoprotein genes protect mice against lethal influenza virus challenge.

    PubMed

    Zhang, H; Tang, X; Zhu, C; Song, Y; Yin, J; Xu, J; Ertl, H C J; Zhou, D

    2015-08-01

    Influenza virus (IV) infection is a major public health problem, causing millions of cases of severe illness and as many as 500 000 deaths each year worldwide. Given the limitations of current prevention or treatment of acute influenza, novel therapies are needed. RNA interference (RNAi) through microRNAs (miRNA) is an emerging technology that can suppress virus replication in vitro and in vivo. Here, we describe a novel strategy for the treatment of infuenza based on RNAi delivered by a replication-defective adenovirus (Ad) vector, derived from chimpanzee serotype 68 (AdC68). Our results showed that artificial miRNAs (amiRNAs) specifically targeting conserved regions of the IV genome could effectively inhibit virus replication in human embryonic kidney 293 cells. Moreover, our results demonstrated that prophylactic treatment with AdC68 expressing amiRNAs directed against M1, M2 or nucleoprotein genes of IV completely protected mice from homologous A/PR8 virus challenge and partially protected the mice from heterologous influenza A virus strains such as H9N2 and H5N1. Collectively, our data demonstrate that amiRNAs targeting the conserved regions of influenza A virus delivered by Ad vectors should be pursued as a novel strategy for prophylaxis of IV infection in humans and animals. PMID:25835311

  11. Novel bat adenoviruses with an extremely large E3 gene.

    PubMed

    Tan, Bing; Yang, Xing-Lou; Ge, Xing-Yi; Peng, Cheng; Zhang, Yun-Zhi; Zhang, Li-Biao; Shi, Zheng-Li

    2016-07-01

    Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs. PMID:27032099

  12. Eliminating established tumor in nu/nu nude mice by a TRAIL-armed oncolytic adenovirus

    PubMed Central

    Dong, Fengqin; Wang, Li; Davis, John J.; Hu, Wenxian; Zhang, Lidong; Guo, Wei; Teraishi, Fuminori; Ji, Lin; Fang, Bingliang

    2006-01-01

    Purpose The tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector. Experimental Design We constructed a TRAIL-expressing oncolytic adenovector designated Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors. Results Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing replication-defective adenovector or an oncolytic adenovector expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all subcutaneous xenograft tumors established from a human non-small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term tumor-free survival. Furthermore, we found no treatment-related toxicity. Conclusions Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy. PMID:16951242

  13. Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

    SciTech Connect

    Ugai, Hideyo; Inabe, Kumiko; Yamasaki, Takahito; Murata, Takehide; Obata, Yuichi; Hamada, Hirofumi; Yokoyama, Kazunari K. . E-mail: kazu@brc.riken.jp

    2005-11-25

    In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.

  14. The Challenge for Gene Therapy: Innate Immune Response to Adenoviruses

    PubMed Central

    Thaci, Bart; Ulasov, Ilya V.; Wainwright, Derek A.; Lesniak, Maciej S.

    2011-01-01

    Adenoviruses are the most commonly used vectors for gene therapy. Despite the promising safety profile demonstrated in clinical trials, the efficacy of using adenoviruses for gene therapy is poor. A major hurdle to adenoviral-mediated gene therapy is the innate immune system. Cell-mediated recognition of viruses via capsid components or nucleic acids has received significant attention, principally thought to be regulated by the toll-like receptors (TLRs). Antiviral innate immune responses are initiated by the infected cell, which activates the interferon (IFN) response to block viral replication, while simultaneously releasing chemokines to attract neutrophils, mononuclear- and natural killer-cells. While the IFN and cellular recruitment pathways are activated and regulated independently of each other, both are required to overcome immune escape mechanisms by adenoviruses. Recent work has shown that the generation of adenoviral vectors lacking specific transcriptionally-active regions decreases immune system activation and increases the chance for immune escape. In this review, we elucidate how adenoviral vector modifications alter the IFN and innate inflammatory pathway response and propose future targets with clinically-translational relevance. PMID:21399236

  15. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  16. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    PubMed Central

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples. PMID:15933012

  17. Nuclear Import of Moloney Murine Leukemia Virus DNA Mediated by Adenovirus Preterminal Protein Is Not Sufficient for Efficient Retroviral Transduction in Nondividing Cells

    PubMed Central

    Lieber, André; Kay, Mark A.; Li, Zong-Yi

    2000-01-01

    Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G1/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration. PMID:10623734

  18. Preclinical Assessment of Viral Vectored and Protein Vaccines Targeting the Duffy-Binding Protein Region II of Plasmodium Vivax

    PubMed Central

    de Cassan, Simone C.; Shakri, A. Rushdi; Llewellyn, David; Elias, Sean C.; Cho, Jee Sun; Goodman, Anna L.; Jin, Jing; Douglas, Alexander D.; Suwanarusk, Rossarin; Nosten, François H.; Rénia, Laurent; Russell, Bruce; Chitnis, Chetan E.; Draper, Simon J.

    2015-01-01

    Malaria vaccine development has largely focused on Plasmodium falciparum; however, a reawakening to the importance of Plasmodium vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII) with the human Duffy antigen receptor for chemokines (DARC) makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5), chimpanzee adenovirus serotype 63 (ChAd63), and modified vaccinia virus Ankara (MVA) vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime or in “mixed-modality” adenovirus prime – protein-in-­adjuvant boost regimes (using a recombinant PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants). Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII, and have recently entered clinical trials, which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans. PMID:26217340

  19. Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers.

    PubMed

    Hoyos, Valentina; Del Bufalo, Francesca; Yagyu, Shigeki; Ando, Miki; Dotti, Gianpietro; Suzuki, Masataka; Bouchier-Hayes, Lisa; Alemany, Ramon; Brenner, Malcolm K

    2015-09-01

    Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC. PMID:26084970

  20. Pseudotyping Serotype 5 Adenovirus with the Fiber from Other Serotypes Uncovers a Key Role of the Fiber Protein in Adenovirus 5-Induced Thrombocytopenia.

    PubMed

    Raddi, Najat; Vigant, Frédéric; Wagner-Ballon, Oriane; Giraudier, Stéphane; Custers, Jerome; Hemmi, Silvio; Benihoud, Karim

    2016-02-01

    Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia. PMID:26757054

  1. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-01-01

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene). PMID:26782515

  2. Hybrid nonviral/viral vector systems for improved piggyBac DNA transposon in vivo delivery.

    PubMed

    Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L

    2015-04-01

    The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR. PMID:25557623

  3. Adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines

    PubMed Central

    Tang, De-chu C; Zhang, Jianfeng; Toro, Haroldo; Shi, Zhongkai; Van Kampen, Kent R

    2009-01-01

    A long-sought goal during the battle against avian influenza is to develop a new generation of vaccines capable of mass immunizing humans as well as poultry (the major source of avian influenza for human infections) in a timely manner. Although administration of the currently licensed influenza vaccine is effective in eliciting protective immunity against seasonal influenza, this approach is associated with a number of insurmountable problems for preventing an avian influenza pandemic. Many of the hurdles may be eliminated by developing new avian influenza vaccines that do not require the propagation of an influenza virus during vaccine production. Replication-competent adenovirus-free adenovirus vectors hold promise as a carrier for influenza virus-free avian influenza vaccines owing to their safety profile and rapid manufacture using cultured suspension cells in a serum-free medium. Simple and efficient mass-immunization protocols, including nasal spray for people and automated in ovo vaccination for poultry, convey another advantage for this class of vaccines. In contrast to parenteral injection of adenovirus vector, the potency of adenovirus-vectored nasal vaccine is not appreciably interfered by pre-existing immunity to adenovirus. PMID:19348562

  4. A New Human DSG2-Transgenic Mouse Model for Studying the Tropism and Pathology of Human Adenoviruses

    PubMed Central

    Wang, Hongjie; Beyer, Ines; Persson, Jonas; Song, Hui; Li, ZongYi; Richter, Maximilian; Cao, Hua; van Rensburg, Ruan; Yao, Xiaoying; Hudkins, Kelly; Yumul, Roma; Zhang, Xiao-Bing; Yu, Mujun; Fender, Pascal; Hemminki, Akseli

    2012-01-01

    We have recently reported that a group of human adenoviruses (HAdVs) uses desmoglein 2 (DSG2) as a receptor for infection. Among these are the widely distributed serotypes HAdV-B3 and HAdV-B7, as well as a newly emerged strain derived from HAdV-B14. These serotypes do not infect rodent cells and could not up until now be studied in small-animal models. We therefore generated transgenic mice containing the human DSG2 locus. These mice expressed human DSG2 (hDSG2) at a level and in a pattern similar to those found for humans and nonhuman primates. As an initial application of hDSG2-transgenic mice, we used a green fluorescent protein (GFP)-expressing HAdV-B3 vector (Ad3-GFP) and studied GFP transgene expression by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry subsequent to intranasal and intravenous virus application. After intranasal application, we found efficient transduction of bronchial and alveolar epithelial cells in hDSG2-transgenic mice. Intravenous Ad3-GFP injection into hDSG2-transgenic mice resulted in hDSG2-dependent transduction of epithelial cells in the intestinal and colon mucosa. Our findings give an explanation for clinical symptoms associated with infection by DSG2-interacting HAdVs and provide a rationale for using Ad3-derived vectors in gene therapy. PMID:22457526

  5. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.

    PubMed Central

    Israel, D I; Kaufman, R J

    1989-01-01

    A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion. Images PMID:2546123

  6. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via “Antigen Capsid-Incorporation” strategy

    PubMed Central

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L.; Derdeyn, Cynthia A.; Matthews, Qiana L.

    2016-01-01

    Adenoviral (Ad) vectors in combination with the “Antigen Capsid-Incorporation” strategy have been applied in developing HIV-1 vaccines, due to the vectors’ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the “Antigen Capsid-Incorporation” strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. PMID:26499044

  7. Adenovirus detection in Guthrie cards from paediatric leukaemia cases and controls

    PubMed Central

    Vasconcelos, G M; Kang, M; Pombo-de-Oliveira, M S; Schiffman, J D; Lorey, F; Buffler, P; Wiemels, J L

    2008-01-01

    Archived neonatal blood cards (Guthrie cards) from children who later contracted leukaemia and matched normal controls were assayed for adenovirus (AdV) C DNA content using two highly sensitive methods. In contrast to a previous report, AdV DNA was not detected at a higher frequency among neonates who later developed leukaemia, when compared with controls. PMID:19002185

  8. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob.

    PubMed

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-03-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 10(4) level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  9. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  10. CCL21/IL21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells.

    PubMed

    Li, Yang; Li, Yi-Fei; Si, Chong-Zhan; Zhu, Yu-Hui; Jin, Yan; Zhu, Tong-Tong; Liu, Ming-Yuan; Liu, Guang-Yao

    2016-07-15

    Multigene-armed oncolytic adenoviruses are capable of efficiently generating a productive antitumor immune response. The chemokine (C-C motif) ligand 21 (CCL21) binds to CCR7 on naïve T cells and dendritic cells (DCs) to promote their chemoattraction to the tumor and resultant antitumor activity. Interleukin 21 (IL21) promotes survival of naïve T cells while maintaining their CCR7 surface expression, which increases their capacity to transmigrate in response to CCL21 chemoattraction. IL21 is also involved in NK cell differentiation and B cell activation and proliferation. The generation of effective antitumor immune responses is a complex process dependent upon coordinated interactions of various subsets of effector cells. Using the AdEasy system, we aimed to construct an oncolytic adenovirus co-expressing CCL21 and IL21 that could selectively replicate in TERTp-positive tumor cells (Ad-CCL21-IL21 virus). The E1A promoter of these oncolytic adenoviruses was replaced by telomerase reverse transcriptase promoter (TERTp). Ad-CCL21-IL21 was constructed from three plasmids, pGTE-IL21, pShuttle-CMV-CCL21 and AdEasy-1 and was homologously recombined and propagated in the Escherichia coli strain BJ5183 and the packaging cell line HEK-293, respectively. Our results showed that our targeted and armed oncolytic adenoviruses Ad-CCL21-IL21 can induce apoptosis in TERTp-positive tumor cells to give rise to viral propagation, in a dose-dependent manner. Importantly, we confirm that these modified oncolytic adenoviruses do not replicate efficiently in normal cells even under high viral loads. Additionally, we investigate the role of Ad-CCL21-IL21 in inducing antitumor activity and tumor specific cytotoxicity of CTLs in vitro. This study suggests that Ad-CCL21-IL21 is a promising targeted tumor-specific oncolytic adenovirus. PMID:27157859

  11. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    PubMed Central

    Gahéry-Ségard, H; Molinier-Frenkel, V; Le Boulaire, C; Saulnier, P; Opolon, P; Lengagne, R; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A; Schatz, C; Courtney, M; Le Chevalier, T; Tursz, T; Guillet, J G; Farace, F

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein. PMID:9410899

  12. Development of replication-competent adenovirus for bladder cancer by controlling adenovirus E1a and E4 gene expression with the survivin promoter

    PubMed Central

    Seo, Ho Kyung; Seo, Jeong Bin; Nam, Jae-Kook; Jeong, Kyung-Chae; Shin, Seung-Pil; Kim, In-Hoo; Lee, Sang Don; Lee, Sang-Jin

    2014-01-01

    Survivin is a member of the inhibitors of apoptosis protein family. Here, we examined survivin expression and confirmed abundant survivin expression in bladder cancer cells. This expression pattern indicated that the transcriptional regulatory elements that control survivin expression could be utilized to discriminate cancer from normal cells. We therefore generated a novel adenovirus termed Ad5/35E1apsurvivinE4 with the following characteristics: 1) E1A and E4 protein expression was dependent on survivin promoter activity; 2) the green fluorescence protein gene was inserted into the genome under the control of the CMV promoter; 3) most of the E3 sequences were deleted, but the construct was still capable of expressing the adenovirus death protein with potent cytotoxic effects; and 4) the fiber knob was from serotype 35 adenovirus. As expected from the abundant survivin expression observed in bladder cancer cells, Ad5/35E1apsurvivinE4 replicated better in cancer cells than in normal cells by a factor of 106 to 102. Likewise, Ad5/35E1apsurvivinE4 exerted greater cytotoxic effects on all bladder cancer cell lines tested. Importantly, Ad5/35E1apsurvivinE4 inhibited the growth of Ku7-Luc orthotopic xenografts in nude mice. Taken together, Ad5/35E1apsurvivinE4 indicates that the survivin promoter may be utilized for the development of a replication-competent adenovirus to target bladder cancers. PMID:25015402

  13. Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

    PubMed Central

    Lin, Baochuan; Vora, Gary J.; Thach, Dzung; Walter, Elizabeth; Metzgar, David; Tibbetts, Clark; Stenger, David A.

    2004-01-01

    The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h. PMID:15243087

  14. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

    PubMed

    Lyons, Mark; Onion, David; Green, Nicky K; Aslan, Kriss; Rajaratnam, Ratna; Bazan-Peregrino, Miriam; Phipps, Sue; Hale, Sarah; Mautner, Vivien; Seymour, Leonard W; Fisher, Kerry D

    2006-07-01

    Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access. PMID:16580883

  15. Adenoviruses in the immunocompromised host.

    PubMed Central

    Hierholzer, J C

    1992-01-01

    Adenoviruses are among the many pathogens and opportunistic agents that cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe, and the serotypes involved are clustered according to the age of the patient and the nature of the immunosuppression. Over 300 adenovirus infections in immunocompromised patients, with an overall case fatality rate of 48%, are reviewed in this paper. Children with severe combined immunodeficiency syndrome and other primary immunodeficiencies are exposed to the serotypes of subgroups B and C that commonly infect young children, and thus their infections are due to types 1 to 7 and 31 of subgenus A. Children with bone marrow and liver transplants often have lung and liver adenovirus infections that are due to an expanded set of subgenus A, B, C, and E serotypes. Adults with kidney transplants have viruses of subgenus B, mostly types 11, 34, and 35, which cause cystitis. This review indicates that 11% of transplant recipients become infected with adenoviruses, with case fatality rates from 60% for bone marrow transplant patients to 18% for renal transplant patients. Patients with AIDS become infected with a diversity of serotypes of all subgenera because their adult age and life-style expose them to many adenoviruses, possibly resulting in antigenically intermediate strains that are not found elsewhere. Interestingly, isolates from the urine of AIDS patients are generally of subgenus B and comprise types 11, 21, 34, 35, and intermediate strains of these types, whereas isolates from stool are of subgenus D and comprise many rare, new, and intermediate strains that are untypeable for practical purposes. It has been estimated that adenoviruses cause active infection in 12% of AIDS patients and that 45% of

  16. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  17. Etoposide enhances antitumor efficacy of MDR1-driven oncolytic adenovirus through autoupregulation of the MDR1 promoter activity.

    PubMed

    Su, Bing-Hua; Shieh, Gia-Shing; Tseng, Yau-Lin; Shiau, Ai-Li; Wu, Chao-Liang

    2015-11-10

    Conditionally replicating adenoviruses (CRAds), or oncolytic adenoviruses, such as E1B55K-deleted adenovirus, are attractive anticancer agents. However, the therapeutic efficacy of E1B55K-deleted adenovirus for refractory solid tumors has been limited. Environmental stress conditions may induce nuclear accumulation of YB-1, which occurs in multidrug-resistant and adenovirus-infected cancer cells. Overexpression and nuclear localization of YB-1 are associated with poor prognosis and tumor recurrence in various cancers. Nuclear YB-1 transactivates the multidrug resistance 1 (MDR1) genes through the Y-box. Here, we developed a novel E1B55K-deleted adenovirus driven by the MDR1 promoter, designed Ad5GS3. We tested the feasibility of using YB-1 to transcriptionally regulate Ad5GS3 replication in cancer cells and thereby to enhance antitumor efficacy. We evaluated synergistic antitumor effects of oncolytic virotherapy in combination with chemotherapy. Our results show that adenovirus E1A induced E2F-1 activity to augment YB-1 expression, which shut down host protein synthesis in cancer cells during adenovirus replication. In cancer cells infected with Ad5WS1, an E1B55K-deleted adenovirus driven by the E1 promoter, E1A enhanced YB-1 expression, and then further phosphorylated Akt, which, in turn, triggered nuclear translocation of YB-1. Ad5GS3 in combination with chemotherapeutic agents facilitated nuclear localization of YB-1 and, in turn, upregulated the MDR1 promoter activity and enhanced Ad5GS3 replication in cancer cells. Thus, E1A, YB-1, and the MDR1 promoter form a positive feedback loop to promote Ad5GS3 replication in cancer cells, and this regulation can be further augmented when chemotherapeutic agents are added. In the in vivo study, Ad5GS3 in combination with etoposide synergistically suppressed tumor growth and prolonged survival in NOD/SCID mice bearing human lung tumor xenografts. More importantly, Ad5GS3 exerted potent oncolytic activity against clinical

  18. Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein.

    PubMed

    Hu, Chao; Zhu, Lihui; Luo, Rong; Dao, Jinwei; Zhao, Jiangping; Shi, Yaojun; Li, Hao; Lu, Ke; Feng, Xingang; Lin, Jiaojiao; Liu, Jinming; Cheng, Guofeng

    2014-11-01

    Schistosomiasis is a worldwide parasitic disease, and while it can be successfully treated with chemotherapy, this does not prevent reinfection with the parasite. Adenovirus vectors have been widely used for vaccine delivery, and a vaccination approach has the potential to prevent infection with Schistosoma. Here, we developed a recombinant adenoviral vector that expresses Schistosoma japonicum inhibitor apoptosis protein (Ad-SjIAP) and assessed its immunoprotective functions against schistosomiasis in mice. Murine immune responses following vaccination were investigated using enzyme-linked immunosorbent assays (ELISA), lymphocyte proliferation, and cytokine assays. The protective immunity in mice was evaluated by challenging with S. japonicum cercariae. Our results indicated that immunization with the Ad-SjIAP in mice induced a strong serum IgG response against IAP including IgG1, IgG2a, and IgG2b. In addition, lymphocyte proliferation experiments showed that mice treated with Ad-SjIAP significantly increased the lymphocyte response upon stimulation with recombinant Schistosoma japonicum inhibitor apoptosis protein (rSjIAP). Moreover, cytokine assays indicated that vaccination of Ad-SjIAP significantly increased the production of interferon (IFN)-γ and IL-2 as compared to the corresponding control group. Furthermore, following the challenge with S. japonicum cercariae, the vaccine conferred moderate protection, with an average rate of 37.95% for worm reduction and 31.7% for egg reduction. Taken together, our preliminarily results suggested that schistosoma IAP may be a potential vaccine against S. japonicum and that adenoviral vectors may serve as an alternative delivery vehicle for schistosome vaccine development. PMID:25185668

  19. Induction of Specific Humoral and Cellular Immune Responses in a Mouse Model following Gene Fusion of HSP70C and Hantaan Virus Gn and S0.7 in an Adenoviral Vector

    PubMed Central

    Li, Kai; Wang, Fang; Zhang, Liang; Ye, Wei; Li, Puyuan; Zhang, Fanglin; Xu, Zhikai

    2014-01-01

    Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV. PMID:24505421

  20. Regulation of adenovirus-mediated elafin transgene expression by bacterial lipopolysaccharide.

    PubMed

    Simpson, A J; Cunningham, G A; Porteous, D J; Haslett, C; Sallenave, J M

    2001-07-20

    Lipopolysaccharide (LPS) is a mediator of inflammatory lung injury. Selective augmentation of host defense molecules such as elafin (an elastase inhibitor with antimicrobial activity) at the onset of pulmonary inflammation is an attractive potential therapeutic strategy. The aim of this study was to determine whether elafin expression could be induced by LPS administered after transfection with adenovirus (Ad) encoding human elafin downstream of the murine cytomegalovirus (CMV) promoter (known to be potentially responsive to LPS). In addition, we aimed to determine the effect of local elafin augmentation on neutrophil migration to the lung. LPS significantly up-regulated elafin expression from pulmonary epithelial cells transfected with Ad-elafin in vitro. In murine airways expression of human elafin was achieved using doses low enough (3 x 10(7) plaque forming units) to circumvent overt vector-induced inflammation. LPS significantly up-regulated human elafin secretion in murine airways treated with Ad-elafin [117 ng/ml in bronchoalveolar lavage fluid (BALF) after LPS administration, 5.9 ng/ml after PBS, p < 0.01)]. Over-expression of elafin significantly augmented LPS-mediated neutrophil migration into the airways in vivo (1.30 x 10(6) neutrophils in BALF after Ad-elafin/LPS treatment, 0.54 x 10(6) after Ad-lacZ/LPS (p < 0.05), 0.63 x 10(6) after PBS/LPS (p < 0.05)) and significantly enhanced human neutrophil migration in vitro. These data suggest novel functions for elafin in neutrophil migration, and that judicious selection of promoters may allow single, low-dose adenoviral administration to effect inflammation-specific expression of potentially therapeutic transgenes. PMID:11485631

  1. Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle

    PubMed Central

    Dulal, Pawan; Wright, Daniel; Ashfield, Rebecca; Hill, Adrian V.S.; Charleston, Bryan; Warimwe, George M.

    2016-01-01

    Development of safe and efficacious vaccines whose potency is unaffected by long-term storage at ambient temperature would obviate major vaccine deployment hurdles and limit wastage associated with breaks in the vaccine cold chain. Here, we evaluated the immunogenicity of a novel chimpanzee adenovirus vectored Rift Valley Fever vaccine (ChAdOx1-GnGc) in cattle, following its thermostabilisation by slow desiccation on glass fiber membranes in the non-reducing sugars trehalose and sucrose. Thermostabilised ChAdOx1-GnGc vaccine stored for 6 months at 25, 37 or 45 °C elicited comparable Rift Valley Fever virus neutralising antibody titres to those elicited by the ‘cold chain’ vaccine (stored at −80 °C throughout) at the same dose, and these were within the range associated with protection against Rift Valley Fever in cattle. The results support the use of sugar-membrane thermostabilised vaccines in target livestock species. PMID:27020712

  2. Carbon Monoxide Prevents Hypertension and Proteinuria in an Adenovirus sFlt-1 Preeclampsia-Like Mouse Model

    PubMed Central

    Venditti, Carolina C.; Casselman, Richard; Young, Iain; Karumanchi, S. Ananth; Smith, Graeme N.

    2014-01-01

    Preeclampsia (PE) remains a leading cause of maternal and neonatal morbidity and mortality worldwide. Smoking cigarettes is associated with a decreased incidence of PE. Based on this observation and previous work, we hypothesize that women who smoke have a lower risk of developing PE because of elevated levels of carbon monoxide (CO) in their blood. The objective of this study was to determine if low-dose CO in ambient air could attenuate the late pregnancy hypertension (HTN) and proteinuria in the Adenovirus (Ad) sFlt-1 PE-like mouse model. Continuous low-dose CO treatment (250 ppm) was started on E10.5 and maintained until E17.5. Compared to control and Ad empty vector, AdsFlt-1 mice displayed late- gestation HTN (E14.5–17.5) (P<0.05), proteinuria (P<0.05) and reduced Bowman's space which were all prevented with CO treatment. Use of the Ad (with/without sFlt-1) or CO had no effect (p>0.05) on litter size, fetal resorption numbers and fetal or placental weights. This study shows that treatment with CO can prevent HTN and proteinuria in a mouse model of PE. It provides a possible mechanism for the reduced incidence of PE in smoking women, and supports the possibility of using CO as a future treatment for PE. PMID:25202912

  3. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  4. In vitro transcription of adenovirus.

    PubMed Central

    Fire, A; Baker, C C; Manley, J L; Ziff, E B; Sharp, P A

    1981-01-01

    A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters. Images PMID:7321101

  5. Inactivation of human adenovirus by sequential disinfection with an alternative UV technology and free chlorine.

    PubMed

    Lee, Jung-Keun; Shin, Gwy-Am

    2011-03-01

    There has been growing concern over human exposure to adenoviruses through drinking water due to the extreme resistance of human adenoviruses to the traditional UV technology (low-pressure (LP) UV). As an effort to develop an effective treatment strategy against human adenoviruses in drinking water, we determined the effectiveness of sequential disinfection with an alternative UV technology (medium-pressure (MP) UV) and free chlorine. Human adenovirus 2 (Ad2) was irradiated with a low dose of MP UV irradiation (10 mJ/cm(2)) through UV collimated apparatus and then exposed to a low dose of free chlorine (0.17 mg/L) at pH 8 and 5°C using a bench-scale chemical disinfection system. A significant inactivation (e.g. 4 log(10)) of Ad2 was achieved with the low doses of MP UV and free chlorine within a very short contact time (∼1.5 min) although there was no apparent synergistic effect on Ad2 between MP UV and free chlorine. Overall, it is likely that the sequential disinfection with UV irradiation and free chlorine should control the contamination of drinking water by human adenoviruses within practical doses of UV and free chlorine typically used in drinking water treatment processes. PMID:21301114

  6. Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure.

    PubMed

    Seth, P

    1994-12-15

    Adenoviruses have been previously shown to enhance the delivery of many ligands including proteins and plasmid DNAs to the cells. The key biochemical step during this process is the ability of adenovirus to disrupt (lyse) the endosome membrane releasing the co-internalized virus and the other ligands into the cytosol (Seth et al, 1986, In: Adenovirus attachment and entry into cells, pp 191-195, American Society for Microbiology, Washington, D.C.). To understand the role of the adenovirus proteins involved in the endosome lysis, it is further shown here that empty capsids of adenovirus also possess this membrane vesicle lytic activity; though the activity is about 5-times lower than the adenovirus. Incubation of adenovirus with low concentration of ionic detergent or brief exposure to 45 degrees C destroyed this lytic activity without affecting the adenovirus binding to cell surface receptor, suggesting the lytic activity of adenovirus to be of enzymatic nature. However, exposing adenovirus to conditions that can disrupt adenovirus capsid structure such as heating at 65 degrees C, treating with 0.5% SDS, treating with different proteases, dialyzing against no glycerol buffer, treating with 6 M urea or with 10% pyridine, and sonication destroyed the adenovirus-associated lytic activity. Results suggest the requirement of an intact capsid structure for adenovirus-mediated lysis of the endosome. PMID:7802664

  7. Combination of oncolytic adenovirus and endostatin inhibits human retinoblastoma in an in vivo mouse model.

    PubMed

    Wang, Huiping; Wei, Fang; Li, Huiming; Ji, Xunda; Li, Shuxia; Chen, Xiafang

    2013-02-01

    There is a critical need for new paradigms in retinoblastoma (RB) treatment that would more efficiently inhibit tumor growth while sparing the vision of patients. Oncolytic adenoviruses with the ability to selectively replicate and kill tumor cells are a promising strategy for cancer gene therapy. Exploration of a novel targeting strategy for RB utilizing combined oncolytic adenovirus and anti-angiogenesis therapy was applied over the course of the current study with positive results. The oncolytic adenoviruses Ad-E2F1 p-E1A and Ad-TERT p-E1 were constructed. The E1 region was regulated by the E2F-1 promoter or the human telomerase reverse transcriptase (hTERT) promoter, respectively. Effects on both replication and promotion of enhanced green fluorescent protein (EGFP) expression were observed in the replication-defective adenovirus Ad-EGFP in diverse cancer cell lines, HXO-RB44, Y79, Hep3B, NCIH460, MCF-7 and HLF. The cancer cell death induced by these agents was also explored. The in situ RB model demonstrated that mice with tumors treated with the oncolytic adenovirus and replication-defective adenovirus Ad-endostatin exhibited notable cancer cell death. This anticancer effect was further examined by stereo microscope, and the survival rate of experimental mice was determined. Both Ad-E2F1 p-E1A and Ad-TERT p-E1 replicated specifically in cancer cells in vitro and promoted EGFP expression in Ad-EGFP, although Ad-E2F1 p-E1A demonstrated superior EGFP promotion activity than Ad-TERT p-E1. In Hep3B, NCIH460 and MCF-7 cells, the number of Ad-TERT p-E1 copies was observed to exceed of the number of Ad-E2F1 p-E1A copies by a minimum of 10-fold. Furthermore, Ad-TERT p-E1 demonstrated significantly superior oncolytic effects in the RB mouse model, and Ad-endostatin effectively suppressed tumor growth and extended the overall lifespan of subjects; however, the Ad-E2F1 p-E1A was clearly less effective in attaining these goals. Most notably, the antitumor effect and

  8. Development of an Ad5H3 Chimera Using the “Antigen Capsid-Incorporation” Strategy for an Alternative Vaccination Approach

    PubMed Central

    Gu, Linlin; Icyuz, Mert; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Johnston, Alison E.; Matthews, Qiana L.

    2016-01-01

    Background: Adenovirus type 5 (Ad5) achieved success as a conventional transgene vaccine vector in preclinical trials, however; achieved poor efficiency in some of the clinical trials, due to the major hurdle associated with Ad5 pre-existing immunity (PEI) in the majority of the human population. Objective: We sought to generate Ad5-based chimeras to assess their capabilities to bypass this bottleneck and to induce antigen-specific humoral immune response. Methods: A His6 tag was incorporated into the hypervariable region 2 (HVR2) of hexon3 (H3) capsid protein using the “Antigen Capsid-Incorporation” strategy. This lead to the construction of a viral chimera, Ad5H3-HVR2-His. Ad5H3 was generated previously by substituting the hexon of Ad5 (hexon5) with the hexon from adenovirus type 3 (Ad3). Results: His6 was presented on the viral capsid surface and recognized by a His6 antibody. An in vitro neutralization assay with Ad5 sera indicated the ability of Ad5 chimeras to partially escape Ad5 immunity. Immunization with Ad5H3-HVR2-His generated significant humoral response to the incorporated tagged peptide, when compared to the immunizations with controls. Conclusion: Based on our in vitro studies the data suggested that Ad5H3 as a novel chimeric vaccine platform yields the possibility to escape Ad5 neutralization, and the potential to generate robust humoral immunity against incorporated antigens using the “Antigen Capsid-Incorporation” strategy. PMID:27335626

  9. Intratracheal Instillation of High Dose Adenoviral Vectors Is Sufficient to Induce Lung Injury and Fibrosis in Mice

    PubMed Central

    Zhou, Qiyuan; Chen, Tianji; Bozkanat, Melike; Ibe, Joyce Christina F.; Christman, John W.; Raj, J. Usha; Zhou, Guofei

    2014-01-01

    Rationale Replication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis. Methods We instilled Ad viruses ranging from 107 to 1.625×109 ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining. Results Instillation of high dose Ad vectors (1.625×109 ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites. Conclusions Our results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner. PMID:25551570

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

    SciTech Connect

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

    2009-12-01

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

  11. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    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. PMID:14970588

  12. [Anti-adenovirus activity of a substance and medical form of ribamydil in cell culture].

    PubMed

    Nosach, L N; Diachenko, N S; Zhovnovataia, V L

    2009-01-01

    The inhibiting effect of ribamydil on adenovirus reproduction was studied under the determination of the number of cells with virus- induced DNA-containing intranucleus inclusion bodies and hexone antigen, the synthesis of adenovirus proteins and the infection virus by t he investigation. EC50 of ribamydil substance is 4-8 microg/ml, but complete suppression of adenovirus genome expression was found when adding ribamydil after the virus adsorption, in concentrations of 125-500 microg/ml. The original effect of ribamydil on the expression of adenovirus genome was found under its effect in concentration of 31 microg/ml. Intranucleus virus-induced inclusion bodies of the early type only were found under these conditions. Synthesis of the structural virus polypeptides, including hexone polypeptide (II) and non-structural polypeptide 100K, taking part in hexone trimerization, proceed intensively but without formation of immunologically active hexone. The inhibiting effect of officinal form of ribamydil was less expressed as compared with the substance (EC50: 62 microg/ml). The work results prove that the therapeutic effect of ribamydil (ribavirin) under treatment of adenovirus infections may be achieved in case when it is used in a dose excluding the expression of the adenovirus genome. PMID:20458939

  13. Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo

    PubMed Central

    Fejer, György; Drechsel, Lisa; Liese, Jan; Schleicher, Ulrike; Ruzsics, Zsolt; Imelli, Nicola; Greber, Urs F.; Keck, Simone; Hildenbrand, Bernd; Krug, Anne; Bogdan, Christian; Freudenberg, Marina A.

    2008-01-01

    The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease. PMID:19008951

  14. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients.

    PubMed

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as "off-the-shelf" therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  15. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    PubMed Central

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  16. Viral vector tropism for supporting cells in the developing murine cochlea.

    PubMed

    Sheffield, Abraham M; Gubbels, Samuel P; Hildebrand, Michael S; Newton, Stephen S; Chiorini, John A; Di Pasquale, Giovanni; Smith, Richard J H

    2011-07-01

    Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear. PMID:21530627

  17. Selective transduction of mature DC in human skin and lymph nodes by CD80/CD86-targeted fiber-modified Adenovirus-5/3

    PubMed Central

    van de Ven, Rieneke; Lindenberg, Jelle J.; Oosterhoff, Dinja; van den Tol, M. Petrousjka; Rosalia, Rodney A.; Murakami, Miho; Everts, Maaike; Scheffer, George L.; Scheper, Rik J.; de Gruijl, Tanja D.; Curiel, David T.

    2009-01-01

    In vivo targeting of dendritic cells (DC) represents an attractive alternative to currently applied ex vivo DC-based genetic tumor vaccination protocols. Finding the optimal vector for in vivo targeting of DC is important for such strategies. We therefore tested a panel of subgroup C/B chimeric and fiber-modified adenoviruses (Ad) for their relative capacity to transduce human DC. We made use of in vitro generated Langerhans Cells (LC) as well as of ex vivo human skin and melanoma-draining lymph node (LN) derived DC. Of the tested viruses the C/B-chimeric Ad5/3 virus most efficiently transduced in vitro generated LC. In addition, Ad5/3 preferentially targeted mature myeloid DC from human skin and draining LN and transduced them at significantly higher frequencies than Ad5. In addition, Ad5/3 was more specific for mature human skin-derived CD1a+ CD83+ DC than the previously reported DC-transducing C/B-chimeric vector Ad5/35, infecting less bystander cells. It was previously reported that Ad5/3 transduced human monocyte-derived DC by binding to the B7 molecules CD80 and CD86. High-efficiency transduction of mature skin-derived DC was similarly shown to be mediated through binding to CD80/CD86 and not to interfere with subsequent T cell priming. We conclude that Ad5/3, in combination with DC-activating adjuvants, represents a promising therapeutic tool for the in vivo transduction of mature DC, and may be less likely to induce unwanted side effects such as immune tolerance through the infection of non-professional antigen-presenting cells. PMID:19816192

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

    PubMed

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

    2016-01-01

    Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy. PMID:26909111

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

    PubMed Central

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

    2016-01-01

    Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy. PMID:26909111

  20. Adenovirus type 7 associated with severe and fatal acute lower respiratory infections in Argentine children

    PubMed Central

    Carballal, Guadalupe; Videla, Cristina; Misirlian, Alicia; Requeijo, Paula V; Aguilar, María del Carmen

    2002-01-01

    Background Adenoviruses are the second most prevalent cause of acute lower respiratory infection of viral origin in children under four years of age in Buenos Aires, Argentina. The purpose of this study was to analyze the clinical features and outcome of acute lower respiratory infection associated with different adenovirus genotypes in children. Methods Twenty-four cases of acute lower respiratory infection and adenovirus diagnosis reported in a pediatric unit during a two-year period were retrospectively reviewed. Adenovirus was detected by antigen detection and isolation in HEp-2 cells. Adenovirus DNA from 17 isolates was studied by restriction enzyme analysis with Bam HI and Sma I. Results Subgenus b was found in 82.3% of the cases, and subgenus c in 17.7%. Within subgenus b, only genotype 7 was detected, with genomic variant 7h in 85.7% (12/14) and genomic variant 7i in 14.3% (2/14). Mean age was 8.8 ±; 6 months, and male to female ratio was 3.8: 1. At admission, pneumonia was observed in 71% of the cases and bronchiolitis in 29%. Malnutrition occurred in 37% of the cases; tachypnea in 79%; chest indrawing in 66%; wheezing in 58%; apneas in 16%; and conjunctivitis in 29%. Blood cultures for bacteria and antigen detection of other respiratory viruses were negative. During hospitalization, fatality rate was 16.7% (4 /24). Of the patients who died, three had Ad 7h and one Ad 7i. Thus, fatality rate for adenovirus type 7 reached 28.6% (4/14). Conclusions These results show the predominance of adenovirus 7 and high lethality associated with the genomic variants 7h and 7i in children hospitalized with acute lower respiratory infection. PMID:12184818

  1. Antitumor activity of adenoviral vector containing T42 and 4xT42 peptide gene through inducing apoptosis of tumor cells and suppressing angiogenesis.

    PubMed

    Zhang, Xiong; Qi, Dong-Dong; Zhang, Ting-Ting; Chen, Qing-Xin; Wang, Guang-Zhi; Sui, Guang-Yu; Hao, Xue-Wei; Sun, Shouli; Song, Xue; Chen, Ying-Li

    2015-03-01

    The T42 peptide, generated from two active fragments of tumstatin, has been shown to have anti‑tumor activity. The adenoviral vector is the most frequently used vector in research and clinical trials for gene therapy. In the present study, the anti‑tumor activity of the T42 peptide and quadruple T42 (4xT42) peptide adenoviral vectors were elucidated for the first time, to the best of our knowledge. Human embryonic kidney 293 cells were infected with plasmid adenovirus (pAd)‑enhanced green fluorescent protein (EGFP)‑T42 or pAd‑EGFP‑4xT42 and the expression of the T42 and 4xT42 genes was confirmed by the identification of GFP expression and reverse transcription polymerase chain reaction experiments. The anti‑cancer effects of pAd‑EGFP‑T42 and pAd‑EGFP‑4xT42 on breast cancer cells in vivo and in vitro were subsequently investigated. The results indicated that the packaging of the recombinant adenoviruses with the viral titer was successful, following purification at 5x109 plaque forming units/ml. The results also revealed that the recombinant adenoviruses promoted apoptosis in MCF‑7 breast cancer cells and inhibited cancer growth. Through the analysis of caspase‑3, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein expression, it was demonstrated that the T42/4xT42 peptide may induce apoptosis via the mitochondrial pathway. In addition, mouse xenograft experiments confirmed that the T42 peptide inhibited tumor growth and reduced angiogenesis in vivo. In conclusion, the results of the present study indicated that the T42 and 4xT42 peptide genes, transfected by a recombinant adenovirus, may provide a potential novel strategy for the treatment of breast cancer. PMID:25384346

  2. Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron

    PubMed Central

    2013-01-01

    Background The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. Results Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. Conclusions Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine. PMID:23767961

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

    PubMed Central

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

    2015-01-01

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

  4. Novel Adenoviruses in Wild Primates: a High Level of Genetic Diversity and Evidence of Zoonotic Transmissions ▿ †

    PubMed Central

    Wevers, Diana; Metzger, Sonja; Babweteera, Fred; Bieberbach, Marc; Boesch, Christophe; Cameron, Kenneth; Couacy-Hymann, Emmanuel; Cranfield, Mike; Gray, Maryke; Harris, Laurie A.; Head, Josephine; Jeffery, Kathryn; Knauf, Sascha; Lankester, Felix; Leendertz, Siv Aina J.; Lonsdorf, Elizabeth; Mugisha, Lawrence; Nitsche, Andreas; Reed, Patricia; Robbins, Martha; Travis, Dominic A.; Zommers, Zinta; Leendertz, Fabian H.; Ehlers, Bernhard

    2011-01-01

    Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events. PMID:21835802

  5. Partial characterization of new adenoviruses found in lizards.

    PubMed

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

    2014-06-01

    In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species. PMID:25000689

  6. Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever

    PubMed Central

    Warimwe, George M.; Gesharisha, Joseph; Carr, B. Veronica; Otieno, Simeon; Otingah, Kennedy; Wright, Danny; Charleston, Bryan; Okoth, Edward; Elena, Lopez-Gil; Lorenzo, Gema; Ayman, El-Behiry; Alharbi, Naif K.; Al-dubaib, Musaad A.; Brun, Alejandro; Gilbert, Sarah C.; Nene, Vishvanath; Hill, Adrian V. S.

    2016-01-01

    Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A ‘One Health’ vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs. PMID:26847478

  7. Three-vector system for high-level functional expression of value-added co-products with xylose isomerase and xylulokinase in an industrial saccharomyces cerevisiae strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease rapidly produces large amounts of soluble functional protein. It provides high levels of expression for three different proteins sim...

  8. Mutation of the fiber shaft heparan sulphate binding site of a 5/3 chimeric adenovirus reduces liver tropism.

    PubMed

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology. PMID:23585829

  9. Mutation of the Fiber Shaft Heparan Sulphate Binding Site of a 5/3 Chimeric Adenovirus Reduces Liver Tropism

    PubMed Central

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver