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Sample records for armed replicating adenoviruses

  1. 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

  2. The Dual Nature of Nek9 in Adenovirus Replication

    PubMed Central

    Jung, Richard; Radko, Sandi

    2015-01-01

    ABSTRACT To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks

  3. 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

  4. 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

  5. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption. PMID:26561828

  6. Oncolytic Replication of E1b-Deleted Adenoviruses.

    PubMed

    Cheng, Pei-Hsin; Wechman, Stephen L; McMasters, Kelly M; Zhou, Heshan Sam

    2015-11-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption. PMID:26561828

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

    PubMed Central

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

    2015-01-01

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents. PMID:26280277

  8. 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.

  9. 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...

  10. 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...

  11. 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...

  12. 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

  13. Adenovirus DNA template for late transcription is not a replicative intermediate.

    PubMed Central

    Brison, O; Kédinger, C; Chambon, P

    1979-01-01

    The relationship between adenovirus replication and late transcription has been investigated using viral replication and transcription complexes isolated from infected HeLa cell nuclei. These two types of complexes extracted from adenovirus type 2-infected cell nuclei did not sediment at the same rate on sucrose gradients. Viral replicative intermediates were quantitatively precipitated by immunoglobulins raised against purified 72,000-dalton DNA-binding protein, whereas viral transcription complexes remained in the supernatant. These results show that late transcription does not occur on active replication complexes or on 72,000-dalton DNA-binding protein-containing replicative intermediates inactive in DNA synthesis. Additional evidence is presented indicating that it is very unlikely that replicative intermediates lacking the 72,000-dalton DNA-binding protein could be the template for late transcription. PMID:232191

  14. 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

  15. Oncolytic Adenoviruses Armed with Thymidine Kinase Can Be Traced by PET Imaging and Show Potent Antitumoural Effects by Ganciclovir Dosing

    PubMed Central

    Abate-Daga, Daniel; Andreu, Nuria; Camacho-Sánchez, Juan; Alemany, Ramon; Herance, Raúl; Millán, Olga; Fillat, Cristina

    2011-01-01

    Replication-competent adenoviruses armed with thymidine kinase (TK) combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET) imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV) induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing. PMID:22028820

  16. Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

    PubMed Central

    Wides, R J; Challberg, M D; Rawlins, D R; Kelly, T J

    1987-01-01

    The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions. Images PMID:3821730

  17. Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication.

    PubMed Central

    Rijnders, A W; van Bergen, B G; van der Vliet, P C; Sussenbach, J S

    1983-01-01

    Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed. Images PMID:6672772

  18. 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

  19. 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...

  20. 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

  1. Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

    PubMed Central

    van Bergen, B G; van der Ley, P A; van Driel, W; van Mansfeld, A D; van der Vliet, P C

    1983-01-01

    Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication. Images PMID:6300787

  2. 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

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

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  4. Enhanced Transduction and Replication of RGD-Fiber Modified Adenovirus in Primary T Cells

    PubMed Central

    Sengupta, Sadhak; Ulasov, Ilya V.; Thaci, Bart; Ahmed, Atique U.; Lesniak, Maciej S.

    2011-01-01

    Background Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45% of splenic T cells were transduced by Ad-RGD. Conclusions Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin. PMID:21464908

  5. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Ran-yi; Zhou, Ling; Zhang, Yan-ling; Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min; Li, Li-xia; Fu, Xiang; Wu, Jiang-xue; Huang, Wenlin

    2013-12-13

    Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

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

    PubMed

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

    2015-03-01

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

  7. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2007-09-01

    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

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

    PubMed

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

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future. PMID:27557721

  9. The adenovirus terminal protein influences binding of replication proteins and changes the origin structure.

    PubMed Central

    Pronk, R; van der Vliet, P C

    1993-01-01

    The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins. Images PMID:8506126

  10. 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

  11. 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

  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. 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

  14. Unique conditionally replication competent bipartite adenoviruses-cancer terminator viruses (CTV): efficacious reagents for cancer gene therapy.

    PubMed

    Sarkar, Devanand; Su, Zao-Zhong; Fisher, Paul B

    2006-07-01

    The frequent resistance of aggressive cancers to currently available therapies, such as radiotherapy and chemotherapy, mandates development of targeted, nontoxic and more efficacious treatment protocols. Conditionally replication competent adenoviruses (CRCAs) that induce oncolysis by cancer-specific replication are currently being evaluated in clinical trials. However, a single modality approach may not be sufficient to completely eradicate cancer in a patient, because most cancers arise from abnormalities in multiple genetic and signal transduction pathways. The promoter region of rodent progression elevated gene-3 (PEG-3), cloned and characterized in our laboratory, embodies the unique property of increased activity in a broad range of tumor cells, both rodent and human, when compared to normal counterparts. Bipartite adenoviruses were engineered to express the E1A gene, necessary for viral replication, under control of the PEG-3 promoter (PEG-Prom) and simultaneously express a second transgene in the E3 region that encodes an apoptosis-inducing and immunomodulatory cytokine, either immune interferon (IFN-gamma) or melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). These conditionally replication competent bipartite adenoviruses, referred to as cancer terminator viruses (CTVs), facilitated cancer-selective adenovirus replication, robust transgene expression and apoptosis induction with complete eradication of both primary and distant (metastatic) human cancers xenotransplanted in athymic nude mice. These findings suggest that CTVs might prove efficacious for the therapy of primary and advanced neoplastic diseases. PMID:16861924

  15. Targeting lung cancer stem-like cells with TRAIL gene armed oncolytic adenovirus

    PubMed Central

    Yang, Yu; Xu, Haineng; Huang, Weidan; Ding, Miao; Xiao, Jing; Yang, Dongmei; Li, Huaguang; Liu, Xin-Yuan; Chu, Liang

    2015-01-01

    Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors-defined serum-free medium. A549 sphere cells displayed CSC properties, including chemo-resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55-EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55-TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55-TRAIL. In the A549 sphere cells xenograft models, ZD55-TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs. PMID:25683371

  16. Immunotherapeutic effects of cytokine-induced killer cells combined with CCL21/IL15 armed oncolytic adenovirus in TERT-positive tumor cells.

    PubMed

    Ye, Jun-Feng; Lin, Yuan-Qiang; Yu, Xiu-Hua; Liu, Ming-Yuan; Li, Yang

    2016-09-01

    The effective antitumor immune responses are dependent on coordinate interaction of various effector cells. Thus, the combination of adoptive immunotherapy and target gene therapy is capable of efficiently generating a productive antitumor immune response. We investigated whether combination of cytokine-induced killer (CIK) cells adoptive immunotherapy and CCL21/IL15 armed oncolytic adenovirus could induce the enhanced antitumor activity. The CCL21/IL15 co-expression oncolytic adenoviruses were constructed by using the AdEasy system, which uses homologous recombination with shuttle plasmids and full length Ad backbones. This conditionally replicating adenoviruses CRAd-CCL21-IL15 could induce apoptosis in TERTp-positive tumor cells for viral propagation, but do not replicate efficiently in normal cells, because the E1A promoter was replaced by telomerase reverse transcriptase promoter (TERTp). Our results showed that the combination of CIK cells and CRAd-CCL21-IL15 could induce higher antitumor activity than either CIK cells or CRAd-CCL21-IL15 alone. This combined treatment could induce the tumor specific cytotoxicity of CTLs (cytotoxic T lymphocytes) in vitro. Moreover, the treatment of established tumors with the combined therapy of CIK cells and CRAd-CCL21-IL15 resulted in tumor regression. This study suggests that the combined treatment by adoptive immunotherapy and gene therapy is a promising strategy for the therapy of tumor. PMID:27380620

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

    PubMed Central

    Komatsu, Tetsuro; Nagata, Kyosuke

    2015-01-01

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

  19. 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

  20. Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

    PubMed Central

    Pettit, S C; Horwitz, M S; Engler, J A

    1988-01-01

    Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP. Images PMID:3336069

  1. Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses for pan-carcinoma application

    PubMed Central

    Takayama, K; Reynolds, PN; Adachi, Y; Kaliberova, L; Uchino, J; Nakanishi, Y; Curiel, DT

    2007-01-01

    Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis. Thus, new therapeutic approaches are needed. Lung tumor formation depends on angiogenesis in which the vascular endothelial growth factor (VEGF) produced by cancer cells plays a pivotal role. Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth; however, the anticancer effect with this therapy is weakened after the intratumoral vascular network is completed. In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells. These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer. Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells. Since tumor-associated angiogenesis via VEGF signalling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment. PMID:17024232

  2. Augmenting the antitumor effect of TRAIL by SOCS3 with double-regulated replicating oncolytic adenovirus in hepatocellular carcinoma.

    PubMed

    Wei, Rui-Cheng; Cao, Xin; Gui, Jing-Hua; Zhou, Xiu-Mei; Zhong, Dan; Yan, Qiao-Lin; Huang, Wei-Dan; Qian, Qi-Jun; Zhao, Feng-Li; Liu, Xin-Yuan

    2011-09-01

    Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy. PMID:21361790

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

    PubMed

    Toth, Karoly; Lee, Sang R; Ying, Baoling; Spencer, Jacqueline F; Tollefson, Ann E; Sagartz, John E; Kong, Il-Keun; Wang, Zhongde; Wold, William S M

    2015-08-01

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

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

    PubMed Central

    Spencer, Jacqueline F.; Tollefson, Ann E.; Sagartz, John E.; Kong, Il-Keun; Wang, Zhongde; Wold, William S. M.

    2015-01-01

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

  5. SUPPRESSION OF VIRAL REPLICATION BY GUANIDINE: A COMPARISON OF HUMAN ADENOVIRUSES AND ENTEROVIRUSES (JOURNAL VERSION)

    EPA Science Inventory

    A comparison was made of the relative sensitivities of laboratory strain human adenoviruses and enteroviruses, and recently isolated human enteroviruses, to the presence of guanidine hydrochloride in cell culture media. The concentration of guanidine hydrochloride used was 100 mi...

  6. 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

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

    PubMed

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

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  8. 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

  9. Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2008-04-25

    Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

  10. Replication of ONYX-015, a Potential Anticancer Adenovirus, Is Independent of p53 Status in Tumor Cells

    PubMed Central

    Rothmann, Thomas; Hengstermann, Arnd; Whitaker, Noel J.; Scheffner, Martin; zur Hausen, Harald

    1998-01-01

    The 55-kDa E1B protein of adenovirus, which binds to and inactivates the tumor suppressor protein p53, is not expressed in the adenoviral mutant termed ONYX-015 (i.e., dl1520). It was reported that the mutant virus due to a deletion in E1B is able to replicate only in cells deficient for wild-type p53. Accordingly, dl1520 is currently being evaluated as a potential tool in the therapy of p53 deficient cancers. In contrast, we report here that dl1520 replicates independently of the p53 status in various tumor cell lines (U87, RKO, A549, H1299, and U373). In addition, the inhibition of p53-mediated transcriptional activation in wild-type p53 containing U2OS cells, by overexpression of a transdominant negative p53 mutant, did not render the cells permissive for dl1520 replication. Finally, we show that, depending on the multiplicity of infection, the deleted virus is able to replicate in and to kill primary human cells. Thus, the molecular basis for the growth differences of dl1520 within different cell types remains to be determined. PMID:9811680

  11. Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer.

    PubMed

    Freytag, Svend O; Barton, Kenneth N; Brown, Stephen L; Narra, Vinod; Zhang, Yingshu; Tyson, Don; Nall, Colleen; Lu, Mei; Ajlouni, Munther; Movsas, Benjamin; Kim, Jae Ho

    2007-09-01

    In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer. PMID:17551507

  12. 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

  13. 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

  14. Transcription factors NFI and NFIII/oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication.

    PubMed Central

    Mul, Y M; Verrijzer, C P; van der Vliet, P C

    1990-01-01

    Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain. Images PMID:2214023

  15. 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

  16. Conserved Arginines of Bovine Adenovirus-3 33K Protein Are Important for Transportin-3 Mediated Transport and Virus Replication

    PubMed Central

    Islam, Azharul; Tikoo, Suresh K.

    2014-01-01

    The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion. PMID:25019945

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

    PubMed

    Mesonero, Alexander; Suarez, David L; van Santen, Edzard; Tang, De-Chu C; Toro, Haroldo

    2011-06-01

    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, antibody persistence, transfer of maternal antibodies (MtAb), and interference between MtAb and active in ovo or mucosal immunization with RCA-free recombinant Ad expressing a codon-optimized AIV H5 HA gene from A/turkey/WI/68 (AdTW68.H5(ck)). Vaccine coverage and intrapotency test repeatability were based on anti-H5 hemagglutination inhibition (HI) antibody levels detected in in ovo vaccinated chickens. Even though egg inoculation of each replicate was performed by individuals with varying expertise and with different vaccine batches, the average vaccine coverage of three replicates was 85%. The intrapotency test repeatability, which considers both positive as well as negative values, varied between 0.69 and 0.71, indicating effective vaccination. Highly pathogenic (HP) AIV challenge of chicken groups vaccinated with increasing vaccine doses showed 90% protection in chickens receiving > or = 10(8) ifu (infectious units)/bird. The protective dose 50% (PD50) was determined to be 10(6.5) ifu. Even vaccinated chickens that did not develop detectable antibody levels were effectively protected against HP AIV challenge. This result is consistent with previous findings ofAd-vector eliciting T lymphocyte responses. Higher vaccine doses significantly reduced viral shedding as determined by AIV RNA concentration in oropharyngeal swabs. Assessment of antibody persistence showed that antibody levels of in ovo immunized chickens continued to increase until 12 wk and started to decline after 18 wk of age. Intramuscular (IM) booster vaccination with the same vaccine at 16 wk of age significantly increased the antibody responses in breeder hens, and these responses were maintained at high

  18. 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.

  19. Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

    PubMed Central

    Diffley, J F; Stillman, B

    1986-01-01

    A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide

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

    SciTech Connect

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

    2013-09-01

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

  1. Biosafety studies of carrier cells infected with a replication-competent adenovirus introduced by IAI.3B promoter

    PubMed Central

    Hamada, Katsuyuki; Shirakawa, Toshiro; Terao, Shuji; Gotoh, Akinobu; Tani, Kenzaburo; Huang, Wenlin

    2014-01-01

    The use of carrier cells infected with oncolytic viruses in cancer gene therapy is an attractive method because it can overcome viral immunogenicity and induce tumor immunity and significant antitumor activity. To enable human clinical trials of this treatment, acute and chronic toxicity tests must first be performed to ensure safety. IAI.3B promoter, oncolytic adenovirus AdE3-IAI.3B introduced by IAI.3B promoter, and A549 carrier cells infected with AdE3-IAI.3B were highly active in cancer cells but not in normal cells. Freeze-thawing increased the antitumor effect of A549 carrier cells by promoting the translocation of oncolytic adenovirus particles from the nucleus to the cytoplasm following the rupture of the nuclear membranes. No deaths or abnormal blood test data resulted from acute toxicity tests conducted in nude mice after a single dose. In chronic toxicity tests in rabbits, there were no serious side effects after eight doses of 1.25 × 107 cells/kg or less for 4 weeks; a significant immune response is known to elicit increased numbers of antiadenovirus antibodies and enlarge the spleen. From these results, it could be concluded that cancer gene therapy of recurrent solid tumors using carrier cells can be safely trialed in humans. PMID:26015963

  2. 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

  3. Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

    PubMed

    Wang, Shuchao; Sun, Chenglong; Zhang, Shoufeng; Zhang, Xiaozhuo; Liu, Ye; Wang, Ying; Zhang, Fei; Wu, Xianfu; Hu, Rongliang

    2015-09-01

    The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines. PMID:26143474

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

    PubMed Central

    Sakurai, Fuminori; Narii, Nobuhiro; Tomita, Kyoko; Togo, Shinsaku; Takahashi, Kazuhisa; Machitani, Mitsuhiro; Tachibana, Masashi; Ouchi, Masaaki; Katagiri, Nobuyoshi; Urata, Yasuo; Fujiwara, Toshiyoshi; Mizuguchi, Hiroyuki

    2016-01-01

    Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)–expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus–adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell–specific microRNA, miR-142-3p, were incorporated into the 3′-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells. PMID:26966699

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

    PubMed

    Sakurai, Fuminori; Narii, Nobuhiro; Tomita, Kyoko; Togo, Shinsaku; Takahashi, Kazuhisa; Machitani, Mitsuhiro; Tachibana, Masashi; Ouchi, Masaaki; Katagiri, Nobuyoshi; Urata, Yasuo; Fujiwara, Toshiyoshi; Mizuguchi, Hiroyuki

    2016-01-01

    Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells. PMID:26966699

  7. 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.

  8. Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells.

    PubMed Central

    van Bergen, B G; van der Vliet, P C

    1983-01-01

    Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation. Images PMID:6302326

  9. [Construction and experimental immunity of recombinant replication-competent canine adenovirus type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus].

    PubMed

    Gao, Yu-Wei; Xia, Xian-Zhu; Wang, Li-Gang; Liu, Dan; Huang, Geng

    2006-04-01

    H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003 (HSN1) HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta EHA and the pPoly2-CAV2 were digested with Nru I /Sal I, respectively. The purified Nru I/Sal I DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA. PMID:16736595

  10. 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

  11. 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

  12. 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

  13. Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

    PubMed

    Yamauchi, Suguru; Kawamura, Kiyoko; Okamoto, Shinya; Morinaga, Takao; Jiang, Yuanyuan; Shingyoji, Masato; Sekine, Ikuo; Kubo, Shuji; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

    2015-12-01

    Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad. PMID:26373551

  14. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  15. 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...

  16. 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

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

    PubMed

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

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors. PMID:27467954

  18. Vaccination with replication-deficient recombinant adenoviruses encoding the main surface antigens of toxoplasma gondii induces immune response and protection against infection in mice.

    PubMed

    Caetano, Bráulia C; Bruña-Romero, Oscar; Fux, Blima; Mendes, Erica A; Penido, Marcus L O; Gazzinelli, Ricardo T

    2006-04-01

    We have generated recombinant adenoviruses encoding three genetically modified surface antigens (SAGs) of the parasite Toxoplasma gondii, that is, AdSAG1, AdSAG2, and AdSAG3. Modifications included the removal of their glycosylphosphatidylinositol (GPI) anchoring motifs and, in some cases, the exchange of the native signal peptide for influenza virus hemagglutinin signal sequence. Adenovirus immunization of BALB/c mice elicited potent antibody responses against each protein, displaying a significant bias toward a helper T cell type 1 (Th1) profile in animals vaccinated with AdSAG1. Furthermore, the presence of parasite-specific IFN-gamma-producing T cells was analyzed by proliferation assays and enzyme-linked immunospot assays in the same animals. Splenocytes from immunized mice secreted IFN-gamma after in vitro stimulation with tachyzoite lysate antigen or with a fraction enriched for membrane-purified GPI-anchored proteins (F3) from the T. gondii tachyzoite surface. Epitopes recognized by CD8+ T cells were identified in SAG1 and SAG3, but not SAG2, sequences, although this protein also induced a specific response. We also tested the capacity of the immune responses detected to protect mice against a challenge with live T. gondii parasites. Although no protection was observed against tachyzoites of the highly virulent RH strain, a significant reduction in cyst loads in the brain was observed in animals challenged with the P-Br strain. Thus, up to 80% of the cysts were eliminated from animals vaccinated with a mixture of the three recombinant viruses. Because adenoviruses seemed capable of inducing Th1-biased protective immune responses against T. gondii antigens, other parasite antigens should be tested alone or in combination with those described here to further develop a protective vaccine against toxoplasmosis. PMID:16610929

  19. 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.

  20. 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.

  1. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs.

    PubMed

    Braucher, Douglas R; Henningson, Jamie N; Loving, Crystal L; Vincent, Amy L; Kim, Eun; Steitz, Julia; Gambotto, Andrea A; Kehrli, Marcus E

    2012-11-01

    Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic losses. To combat IAV infection, the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines, using a prime-boost strategy. These vaccines can provide sterilizing immunity toward homologous virus but often have limited efficacy against a heterologous infection. There is a need for vaccine platforms that induce mucosal and cell-mediated immunity that is cross-reactive to heterologous viruses and can be produced in a short time frame. Nonreplicating adenovirus 5 vector (Ad5) vaccines are one option, as they can be produced rapidly and given intranasally to induce local immunity. Thus, we compared the immunogenicity and efficacy of a single intranasal dose of an Ad5-vectored hemagglutinin (Ad5-HA) vaccine to those of a traditional intramuscular administration of WIV vaccine. Ad5-HA vaccination induced a mucosal IgA response toward homologous IAV and primed an antigen-specific gamma interferon (IFN-γ) response against both challenge viruses. The Ad5-HA vaccine provided protective immunity to homologous challenge and partial protection against heterologous challenge, unlike the WIV vaccine. Nasal shedding was significantly reduced and virus was cleared from the lung by day 5 postinfection following heterologous challenge of Ad5-HA-vaccinated pigs. However, the WIV-vaccinated pigs displayed vaccine-associated enhanced respiratory disease (VAERD) following heterologous challenge, characterized by enhanced macroscopic lung lesions. This study demonstrates that a single intranasal vaccination with an Ad5-HA construct can provide complete protection from homologous challenge and partial protection from heterologous challenge, as opposed to VAERD, which can occur with adjuvanted WIV vaccines. PMID:22933397

  2. The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

    PubMed Central

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

    2011-01-01

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected. PMID:21379379

  3. 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

  4. Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Recombinant Priming and Envelope Protein Boosting Elicits Localized, Mucosal IgA Immunity in Rhesus Macaques Correlated with Delayed Acquisition following a Repeated Low-Dose Rectal SIVmac251 Challenge

    PubMed Central

    Xiao, Peng; Patterson, L. Jean; Kuate, Seraphin; Brocca-Cofano, Egidio; Thomas, Michael A.; Venzon, David; Zhao, Jun; DiPasquale, Janet; Fenizia, Claudio; Lee, Eun Mi; Kalisz, Irene; Kalyanaraman, Vaniambadi S.; Pal, Ranajit; Montefiori, David; Keele, Brandon F.

    2012-01-01

    We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIVmac251 at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the

  5. 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

  6. 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.

  7. Characteristics of Noncultivable Adenoviruses Associated with Diarrhea in Infants: A New Subgroup of Human Adenoviruses

    PubMed Central

    Gary, G. William; Hierholzer, John C.; Black, Robert E.

    1979-01-01

    Virus particles morphologically resembling adenovirus were found in fecal specimens from infants and were examined for cultivability with standard cell culture techniques and for characteristics of human adenoviruses. Specimens from 13 of 15 infants could not be cultivated in cell cultures. The two adenoviruses that were cultivated, types 1 and 31, reacted in the expected manner in all tests. Counterimmunoelectrophoresis with group-specific anti-hexon serum confirmed that the observed particles in the 15 specimens were human adenoviruses. The buoyant density in sucrose of five of the noncultivable adenoviruses in original stool suspensions averaged 1.335 g/cm3 and that of the two cultivable ones averaged 1.332 g/cm3; both groups had typical adenovirus morphology by electron microscopy. Treatment of the specimens and of a variety of tissue culture cells with proteolytic and other enzymes did not improve cultivability. Examination of partially purified virus by immunoelectron microscopy did not reveal evidence of immunoglobulin A, G, or M coating on the particles, an indication that coproantibody inhibition was not the cause of noncultivability. Fluorescent-antibody studies with an antihexon conjugate and counterimmunoelectrophoresis studies of serially passaged noncultivable viruses indicated that the viruses are infecting cells but are not undergoing effective replication. Antisera to three of the noncultivable viruses demonstrated homologous reactions in counterimmunoelectrophoresis with the respective immunizing antigens but showed only low levels of hemagglutination-inhibiting and neutralizing activity to a few of the known human adenoviruses. We concluded that the noncultivable viruses in these infant diarrhea cases were indeed human adenoviruses, were not defective particles, were not bound to coproantibody, were infectious but incapable of effective relication in conventional cell cultures, were serologically related to types 11, 17, 32, and 33, and should be

  8. 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

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

    PubMed Central

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

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses. PMID:27119101

  10. An Infection-enhanced Oncolytic Adenovirus Secreting H. pylori Neutrophil-activating Protein with Therapeutic Effects on Neuroendocrine Tumors

    PubMed Central

    Ramachandran, Mohanraj; Yu, Di; Wanders, Alkwin; Essand, Magnus; Eriksson, Fredrik

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. HP-NAP can mediate antitumor effects by recruiting neutrophils and inducing Th1-type differentiation in the tumor microenvironment. It therefore holds strong potential as a therapeutic gene. Here, we armed a replication-selective, infection-enhanced adenovirus with secretory HP-NAP, Ad5PTDf35-[Δ24-sNAP], and evaluated its therapeutic efficacy against neuroendocrine tumors. We observed that it could specifically infect and eradicate a wide range of tumor cells lines from different origin in vitro. Insertion of secretory HP-NAP did not affect the stability or replicative capacity of the virus and infected tumor cells could efficiently secrete HP-NAP. Intratumoral administration of the virus in nude mice xenografted with neuroendocrine tumors improved median survival. Evidence of biological HP-NAP activity was observed 24 hours after treatment with neutrophil infiltration in tumors and an increase of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and MIP2-α in the systemic circulation. Furthermore, evidence of Th1-type immune polarization was observed as a result of increase in IL-12/23 p40 cytokine concentrations 72 hours postvirus administration. Our observations suggest that HP-NAP can serve as a potent immunomodulator in promoting antitumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus. PMID:23817216

  11. 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

  12. 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.

  13. Neural stem cell-mediated delivery of oncolytic adenovirus.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Qian, Shuo; Spencer, Drew A; Ahmed, Atique U; Lesniak, Maciej S

    2015-01-01

    The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs. PMID:25827347

  14. 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

  15. Interactions of minute virus of mice and adenovirus with host nucleoli.

    PubMed Central

    Walton, T H; Moen, P T; Fox, E; Bodnar, J W

    1989-01-01

    Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation. Images PMID:2760977

  16. 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.

  17. 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.

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

    PubMed Central

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

    2011-01-01

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

  19. Subgenomic viral DNA species synthesized in simian cells by human and simian adenoviruses.

    PubMed Central

    Daniell, E

    1981-01-01

    DNA synthesized after infection of simian tissue culture cells (BSC-1 or CV-1) with human adenovirus type 2 or 5 or with simian adenovirus 7 was characterized. It was demonstrated that as much as 40% of the virus-specific DNA in nuclei of infected monkey cells consists of subgenomic pieces. No subgenomic viral DNA species were detected in the nuclei of human (HeLa) cells infected with these adenovirus types. Restriction analysis showed that these short viral DNA molecules contain normal amounts of the sequences from the ends of the viral genome, whereas internal regions are underrepresented. The production of subgenomic DNAs is not correlated with semipermissive infection. Although adenovirus types 2 and 5 are restricted in monkey cells, these cells are fully permissive for simian adenovirus 7. HR404, an adenovirus type 5 mutant which is not restricted in monkey cells, produced the same percentage of subgenomic DNAs as did its wild type (restricted) parent, and coinfection of monkey cells with adenovirus type 5 DNAs. The array of predominant size classes among the heterogeneously sized short DNAs is serotype specific. Extensive plaque purification and comparison of wild-type adenovirus type 5 with several viral mutants indicated that the distribution of aberrant sizes of DNA is characteristic of the virus and not a result of random replicative errors and then enrichment of particular species. Images PMID:6261009

  20. 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

  1. 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.

  2. 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

  3. Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

    PubMed Central

    Lu, YiQing; Kucharski, Thomas J.; Gamache, Isabelle; Blanchette, Paola; Branton, Philip E.

    2014-01-01

    ABSTRACT Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4−) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4− virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCE Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and

  4. EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

    PubMed Central

    Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

    2013-01-01

    We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

  5. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells

    SciTech Connect

    Zorn, G.A.; Anderson, C.W.

    1981-02-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide.

  6. Oncolytic poxvirus armed with Fas ligand leads to induction of cellular Fas receptor and selective viral replication in FasR-negative cancer.

    PubMed

    Sathaiah, M; Thirunavukkarasu, P; O'Malley, M E; Kavanagh, M A; Ravindranathan, R; Austin, F; Guo, Z S; Bartlett, D L

    2012-03-01

    Tumor necrosis factor superfamily members, including Fas ligand and TRAIL, have been studied extensively for cancer therapy, including as components of gene therapy. We examined the use of FasL expression to achieve tumor-selective replication of an oncolytic poxvirus (vFasL), and explored its biology and therapeutic efficacy for FasR- and FasR+ cancers. Infection of FasR+ normal and MC38 cancer cells by vFasL led to abortive viral replication owing to acute apoptosis and subsequently showed both reduced pathogenicity in non-tumor-bearing mice and reduced efficacy in FasR+ tumor-bearing mice. Infection of FasR- B16 cancer cells by vFasL led to efficient viral replication, followed by late induction of FasR and subsequent apoptosis. Treatment with vFasL as compared with its parental virus (vJS6) led to increased tumor regression and prolonged survival of mice with FasR- cancer (B16) but not with FasR+ cancer (MC38). The delayed induction of FasR by viral infection in FasR- cells provides for potential increased efficacy beyond the limit of the direct oncolytic effect. FasR induction provides one mechanism for tumor-selective replication of oncolytic poxviruses in FasR- cancers with enhanced safety. The overall result is both a safer and more effective oncolytic virus for FasR- cancer. PMID:22116377

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

    PubMed

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

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions. PMID:25629161

  8. Oral Priming with Replicating Adenovirus Serotype 4 Followed by Subunit H5N1 Vaccine Boost Promotes Antibody Affinity Maturation and Expands H5N1 Cross-Clade Neutralization

    PubMed Central

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

    2015-01-01

    A Phase I trial conducted in 2009–2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions. PMID:25629161

  9. Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

    PubMed Central

    Mok, M; Maderious, A; Chen-Kiang, S

    1984-01-01

    We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication. Images PMID:6209554

  10. Neural stem cell-mediated delivery of oncolytic adenovirus

    PubMed Central

    Kim, Julius W.; Kane, J. Robert; Young, Jacob S.; Chang, Alan L.; Kanojia, Deepak; Qian, Shuo; Spencer, Drew A.; Ahmed, Atique U.; Lesniak, Maciej S.

    2015-01-01

    The use of stem cells (SCs) as carriers for therapeutic agents has by now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that could provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and, then, delivered to the tumor, upon which they may evoke pronounced therapeutic efficacy in the animal (Ahmed et al., 2011; Ahmed et al., 2012; Thaci et al., 2012; Tobias et al., 2013). Here in this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs. PMID:25827347

  11. Synthesis of type 2 Adenovirus DNA in the Presence of Cycloheximide

    PubMed Central

    Horwitz, Marshall S.; Brayton, Carol; Baum, Stephen G.

    1973-01-01

    Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur. PMID:4349494

  12. 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...

  13. A novel Golgi protein (GOLPH2)-regulated oncolytic adenovirus exhibits potent antitumor efficacy in hepatocellular carcinoma

    PubMed Central

    Wang, Yigang; Zhao, Hongfang; Zhang, Rong; Ma, Buyun; Chen, Kan; Huang, Fang; Zhou, Xiumei; Cui, Caixia; Liu, Xinyuan

    2015-01-01

    Golgi apparatus is the organelle mainly functioning as protein processing and secretion. GOLPH2 is a resident Golgi glycoprotein, usually called GP73. Recent data displayed that GOLPH2 is a superb hepatocellular carcinoma (HCC) marker candidate, and even its specificity is better than liver cancer marker AFP. Oncolytic adenoviruses are broadly used for targeting cancer therapy due to their selective tumor-killing effect. However, it was reported that traditionally oncolytic adenovirus lack the HCC specificity. In this study, a novel dual-regulated oncolytic adenovirus GD55 targeting HCC was first constructed based on our cancer targeted gene-viral therapeutic strategy. To verify the targeting and effectiveness of GOLPH2-regulated oncolytic adenovirus GD55 in HCC, the anticancer capacity was investigated in HCC cell lines and animal model. The results proved that the novel GOLPH2-regulated GD55 conferred higher adenovirus replication and infectivity for liver cancer cells than oncolytic adenovirus ZD55. The GOLPH2-regulated GD55 exerted a significant grow-suppressing effect on HCC cells in vitro but little damage to normal liver cells. In animal experiment, antitumor effect of GD55 was more effective in HCC xenograft of nude mice than that of ZD55. Thus GOLPH2-regulated GD55 may be a promising oncolytic virus agent for future liver cancer treatment. PMID:25980438

  14. Host cell autophagy modulates early stages of adenovirus infections in airway epithelial cells.

    PubMed

    Zeng, Xuehuo; Carlin, Cathleen R

    2013-02-01

    Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections. PMID:23236070

  15. 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

  16. 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

  17. 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

  18. Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter

    SciTech Connect

    Seiberg, M.; Aloni, Y. ); Levine, A.J. )

    1989-09-01

    Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuator RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.

  19. 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

  20. 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

  1. 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

  2. Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

    PubMed Central

    Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-01

    In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

  3. 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

  4. Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery.

    PubMed

    Rojas, Luis Alfonso; Condezo, Gabriela N; Moreno, Rafael; Fajardo, Carlos Alberto; Arias-Badia, Marcel; San Martín, Carmen; Alemany, Ramon

    2016-09-10

    Recombinant adenoviruses are used as vaccines, gene therapy vectors, and oncolytic viruses. However, the efficacy of such therapies is limited by pre-existing neutralizing antibodies (NAbs), especially when the virus is administered systemically for a wider biodistribution or to reach multiple metastases. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin and maintain the infectivity and replication capacity in presence of NAbs. In pre-immunized mice non-modified viruses are completely neutralized, whereas ABD-modified viruses preserve the ability to transduce target organs, induce oncolysis, or generate immune responses to expressed proteins. Our results indicate that albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus for gene therapy, cancer virotherapy, and vaccination. PMID:27388756

  5. Hyaluronidase Expression by an Oncolytic Adenovirus Enhances Its Intratumoral Spread and Suppresses Tumor Growth

    PubMed Central

    Guedan, Sonia; Rojas, Juan José; Gros, Alena; Mercade, Elena; Cascallo, Manel; Alemany, Ramon

    2010-01-01

    Successful virotherapy requires efficient virus spread within tumors. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix (ECM), could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in mice-bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (MLP) (AdwtRGD-PH20). Intratumoral treatment of human melanoma xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan (HA), enhanced viral distribution, and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus that selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR17) was compared to that of the parental virus ICOVIR15. ICOVIR17 showed more antitumor efficacy following intratumoral and systemic administration in mice with prestablished tumors, along with an improved spread of the virus within the tumor. Importantly, a single intravenous dose of ICOVIR17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR17 is a promising candidate for clinical testing. PMID:20442708

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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...

  11. MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

    PubMed Central

    Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

    2015-01-01

    Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

  12. [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

  13. 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

  14. Safety evaluation of adenovirus type 4 and type 7 vaccine live, oral in military recruits.

    PubMed

    Choudhry, Azhar; Mathena, Julie; Albano, Jessica D; Yacovone, Margaret; Collins, Limone

    2016-08-31

    Before the widespread adoption of vaccination, adenovirus type 4 and type 7 were long associated with respiratory illnesses among military recruits. When supplies were depleted and vaccination was suspended in 1999 for approximately a decade, respiratory illnesses due to adenovirus infections resurged. In March 2011, a new live, oral adenovirus vaccine was licensed by the US Food and Drug Administration and was first universally administered to military recruits in October 2011, leading to rapid, dramatic elimination of the disease within a few months. As part of licensure, a postmarketing study (Sentinel Surveillance Plan) was performed to detect potential safety signals within 42days after immunization of military recruits. This study retrospectively evaluated possible adverse events related to vaccination using data from the Armed Forces Health Surveillance Branch Defense Medical Surveillance System (DMSS) database. Among 100,000 recruits who received the adenovirus vaccine, no statistically significant greater risk of prespecified medical events was observed within 42days after vaccination when compared with a historical cohort of 100,000 unvaccinated recruits. In an initial statistical analysis of International Classification of Disease, 9th Revision, Clinical Modification codes, a statistically significant higher risk for 19 other (not prespecified) medical events occurring in 5 or more recruits was observed among vaccinated compared with unvaccinated groups. After case record data abstraction for attribution and validation, two events (psoriasis [21 vs 7 cases] and serum reactions [12 vs 4 cases]) occurred more frequently in the vaccinated cohort. A causal relation of these rare events with adenovirus vaccination could not be established given confounding factors in the DMSS, such as coadministration of other vaccines and incomplete or inaccurate medical information, for some recruits. Prospective surveillance assessing these uncommon, but potentially

  15. 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

  16. 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

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

    SciTech Connect

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

    2014-11-19

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

  18. Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

    PubMed

    Bramante, Simona; Kaufmann, Johanna K; Veckman, Ville; Liikanen, Ilkka; Nettelbeck, Dirk M; Hemminki, Otto; Vassilev, Lotta; Cerullo, Vincenzo; Oksanen, Minna; Heiskanen, Raita; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Matikainen, Sampsa; Vähä-Koskela, Markus; Koski, Anniina; Hemminki, Akseli

    2015-10-01

    Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma. PMID:25821063

  19. 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

  20. Use of macrophages to target therapeutic adenovirus to human prostate tumors.

    PubMed

    Muthana, Munitta; Giannoudis, Athina; Scott, Simon D; Fang, Hsin-Yu; Coffelt, Seth B; Morrow, Fiona J; Murdoch, Craig; Burton, Julian; Cross, Neil; Burke, Bernard; Mistry, Roshna; Hamdy, Freddie; Brown, Nicola J; Georgopoulos, Lindsay; Hoskin, Peter; Essand, Magnus; Lewis, Claire E; Maitland, Norman J

    2011-03-01

    New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells. PMID:21233334

  1. Replicating vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early work on fish immunology and disease resistance demonstrated fish (like animals and humans) that survived infection were typically resistant to re-infection with the same pathogen. The concepts of resistance upon reinfection lead to the research and development of replicating (live) vaccines in...

  2. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  3. [Quality control of recombinant oncolytic adenovirus/p53].

    PubMed

    Gao, Kai; Bi, Hua; Ding, You-Xue; Li, Yong-Hong; Han, Chun-Mei; Guo, Ying; Rao, Chun-Ming

    2011-12-01

    To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products. PMID:22375422

  4. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

    Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adenoviruses one of (if not the) best models for studying viral evolution on a larger time scale. Phylogenetic calculation can infer the evolutionary distance among adenovirus strains on serotype, species, and genus levels, thus helping the establishment of a correct taxonomy on the one hand, and speeding up the process of typing new isolates on the other. Initially, four major lineages corresponding to four genera were recognized. Later, the demarcation criteria of lower taxon levels, such as species or types, could also be defined with phylogenetic calculations. A limited number of possible host switches have been hypothesized and convincingly supported. Application of the web-based BLAST and MultAlin programs and the freely available PHYLIP package, along with the TreeView program, enables everyone to make correct calculations. In addition to step-by-step instruction on how to perform phylogenetic analysis, critical points where typical mistakes or misinterpretation of the results might occur will be identified and hints for their avoidance will be provided. PMID:17656792

  5. Anti-Viral Drugs for Human Adenoviruses

    PubMed Central

    Waye, Mary Miu Yee; Sing, Chor Wing

    2010-01-01

    There are many stages in the development of a new drug for viral infection and such processes are even further complicated for adenovirus by the fact that there are at least 51 serotypes, forming six distinct groups (A–F), with different degree of infectivity. This review attempts to address the importance of developing pharmaceuticals for adenovirus and also review recent development in drug discovery for adenovirus, including newer strategies such as microRNA approaches. Different drug screening strategies will also be discussed.

  6. Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

    PubMed

    Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

    2011-09-01

    Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC. PMID:21361787

  7. Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

    PubMed Central

    Aparicio, Oscar; Razquin, Nerea; Zaratiegui, Mikel; Narvaiza, Iñigo; Fortes, Puri

    2006-01-01

    Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression. PMID:16415015

  8. An outbreak of adenovirus keratoconjunctivitis in bristol.

    PubMed Central

    Tullo, A B; Higgins, P G

    1979-01-01

    Nineteen cases of keratoconjunctivitis caused by an adenovirus serologically related to types 10 and 19 are described. Seventeen of the patients presented over a period of 7 weeks and included 4 who were involved in a minor outbreak at a factory. The presentation and clinical features closely resembled those caused by adenoviruses types 8 and 19. Mild to severe follicular conjunctivitis, superficial punctate keratitis, discrete subepithelial opacities, membrane formation, and conjunctival scarring were all observed. Images PMID:226115

  9. Antitumor Effects of Oncolytic Adenovirus-Carrying siRNA Targeting Potential Oncogene EphA3

    PubMed Central

    Zhao, Yali; Li, Hailiang; Wu, Ruiqin; Li, Shanhu; Wang, Peng; Wang, Hongtao; Wang, Jian; Zhou, Jianguang

    2015-01-01

    Conditionally replicating adenoviruses (CRAds) armed with antitumor transgenes hold promise for cancer treatment. In previous studies, we showed that the 1504-siRNA targeting potential oncogene EphA3 was an efficient therapeutic transgene and that the telomerase reverse transcriptase promoter (TERTp) driving the CRAd was a more advanced generation of CRAd. Therefore, we combined Ad-TERTp-E1A-1504 by inserting 1504-siRNA into the CRAd to study its antitumor effects and mechanism of action, using Ad-TERTp-E1A-NC and nonreplicating adenovirus carrying 1504-siRNA as controls. Cell viability assays and ED50 studies of growth inhibition confirmed that Ad-TERTp-E1A-1504 has 3.5- and 1,400-fold greater ability to kill EphA3- and TERT-expressing tumor cells compared to Ad-TERTp-E1A-NC and Ad-ΔE1A-1504, respectively. Also, Ad-TERTp-E1A-1504 had little effect on cells that modestly expressed EphA3 and TERT such as 2BS. The antitumor efficacy of Ad-TERTp-E1A-1504 was also validated in vivo. Furthermore, the virus yield of Ad-TERTp-E1A-1504 in C4-2B was ~1,000 times greater than that in 2BS. No obvious differences were observed between Ad-TERTp-E1A-1504 and Ad-TERTp-E1A-NC. Both acridine orange staining and Beclin1 protein measurements indicated that autophagy with Ad-TERTp-E1A-1504 at 5 and 10 MOI was higher than that of Ad-TERTp-E1A-NC. Finally, the classical negatively regulated autophagy signaling pathway, PI3K/AKT/mTOR, was suppressed (reduced phosphorylated form) in contrast to NC, and that this was mediated by 1504-siRNA. Thus, Ad- TERTp-E1A-1504 does not harm normal cells but has dual inhibiting and killing effects on TERT- and EphA3-positive tumor cells, and this effect is mediated by the AKT/mTOR signaling pathway via induction of autophagy. These data may offer a foundation for novel antitumor therapies targeting this mechanism. PMID:25978371

  10. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    SciTech Connect

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  11. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  12. Randomized, placebo-controlled trial to assess the safety and immunogenicity of an adenovirus type 35-based circumsporozoite malaria vaccine in healthy adults.

    PubMed

    Creech, C Buddy; Dekker, Cornelia L; Ho, Dora; Phillips, Shanda; Mackey, Sally; Murray-Krezan, Cristina; Grazia Pau, Maria; Hendriks, Jenny; Brown, Valerie; Dally, Leonard G; Versteege, Isabella; Edwards, Kathryn M

    2013-12-01

    Malaria results in over 650,000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy. PMID:23955431

  13. Randomized, placebo-controlled trial to assess the safety and immunogenicity of an adenovirus type 35-based circumsporozoite malaria vaccine in healthy adults

    PubMed Central

    Creech, C Buddy; Dekker, Cornelia L; Ho, Dora; Phillips, Shanda; Mackey, Sally; Murray-Krezan, Cristina; Grazia Pau, Maria; Hendriks, Jenny; Brown, Valerie; Dally, Leonard G; Versteege, Isabella; Edwards, Kathryn M

    2013-01-01

    Malaria results in over 650 000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 108, 109, 1010, or 1011 vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (1010 and 1011 vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 1011 vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (1010 and 1011 vp/mL). Reactogenicity findings were more common after the 1011 vp/mL dose, although most were mild or moderate in nature and resolved without therapy. PMID:23955431

  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. 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.

  16. human adenoviruses role in ophthalmic pterygium formation

    PubMed Central

    Kelishadi, Mishar; Kelishadi, Mandana; Moradi, Abdolvahab; Javid, Naeme; Bazouri, Masoud; Tabarraei, Alijan

    2015-01-01

    Background: Ophthalmic pterygium is a common benign lesion of unknown origin and the pathogenesis might be vision-threatening. This problem is often associated with exposure to solar light. Recent evidence suggests that potentially oncogenic viruses such as human papillomavirus and Epstein-Barr virus may be involved in the pathogenesis of pterygia. Expression of specific adenovirus genes such as E1A and E1B, which potentially have many functions, may contribute to their oncogenic activity as well as relevance to cellular immortalization. Objectives: For the first time, we aimed to investigate involvement of adenoviruses in pterygium formation. Patients and Methods: Fifty tissue specimens of pterygium from patients undergoing pterygium surgery (as cases), 50 conjunctival swab samples from the same patients and 10 conjunctival biopsy specimens from individuals without pterygium such as patients undergoing cataract surgery (as controls) were analyzed for evidence of adenovirus infection with polymerase chain reaction using specific primers chosen from the moderately conserved region of the hexon gene. Furthermore, β-globin primers were used to access the quality of extracted DNA. Data was analyzed using SPSS (version 16) software. Results: Of 50 patients, 20 were men and 30 women with mean age of 61.1 ± 16.9 years ranged between 22 and 85 years. All samples of pterygia had positive results for adenoviruses DNA with polymerase chain reaction, but none of the negative control groups displayed adenoviruses. The pterygium group and the control groups were β-globin positive. Direct sequencing of PCR products confirmed Adenovirus infection. Conclusions: Adenoviruses might act as a possible cause of pterygium formation and other factors could play a synergistic role in the development. However, further larger studies are required to confirm this hypothesis. PMID:26034543

  17. 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...

  18. 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 ...

  19. Simian adenovirus type 35 has a recombinant genome comprising human and simian adenovirus sequences, which predicts its potential emergence as a human respiratory pathogen

    PubMed Central

    Dehghan, Shoaleh; Seto, Jason; Jones, Morris S.; Dyer, David W.; Chodosh, James; Seto, Donald

    2013-01-01

    Emergent human and simian adenoviruses (HAdVs) may arise from genome recombination. Computational analysis of SAdV type 35 reveals a genome comprising a chassis with elements mostly from two simian adenoviruses, SAdV-B21 and -B27, and regions of high sequence similarity shared with HAdV-B21 and HAdV-B16. Although recombination direction cannot be determined, the presence of these regions suggests prior infections of humans by an ancestor of SAdV-B35, and/or vice versa. Absence of this virus in humans may reflect non-optimal conditions for zoonosis. The presence of both a critical viral replication element found in HAdV genomes and genes that are highly similar to ones in HAdVs suggest the potential to establish in a human host. This allows a prediction that this virus may be a nascent human respiratory pathogen. The recombination potential of human and simian adenovirus genomes should be considered in the use of SAdVs as vectors for gene delivery in humans. PMID:24210123

  20. A Novel Psittacine Adenovirus Identified During an Outbreak of Avian Chlamydiosis and Human Psittacosis: Zoonosis Associated with Virus-Bacterium Coinfection in Birds

    PubMed Central

    Chan, Wan-Mui; Choi, Garnet K. Y.; Zhang, Anna J. X.; Sridhar, Siddharth; Wong, Sally C. Y.; Chan, Jasper F. W.; Chan, Andy S. F.; Woo, Patrick C. Y.; Lau, Susanna K. P.; Lo, Janice Y. C.; Chan, Kwok-Hung; Cheng, Vincent C. C.; Yuen, Kwok-Yung

    2014-01-01

    Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3′ end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3–54.0% for the DNA polymerase, 64.6–70.7% for the penton protein, and 66.1–74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should

  1. A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

    PubMed

    To, Kelvin K W; Tse, Herman; Chan, Wan-Mui; Choi, Garnet K Y; Zhang, Anna J X; Sridhar, Siddharth; Wong, Sally C Y; Chan, Jasper F W; Chan, Andy S F; Woo, Patrick C Y; Lau, Susanna K P; Lo, Janice Y C; Chan, Kwok-Hung; Cheng, Vincent C C; Yuen, Kwok-Yung

    2014-12-01

    Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be

  2. 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

  3. 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

  4. HUMAN ADENOVIRUS TYPE 37 AND THE BALB/C MOUSE: PROGRESS TOWARD A RESTRICTED ADENOVIRUS KERATITIS MODEL (AN AMERICAN OPHTHALMOLOGICAL SOCIETY THESIS)

    PubMed Central

    Chodosh, James

    2006-01-01

    Purpose To establish a mouse model of adenovirus keratitis in order to study innate immune mechanisms in the adenovirus-infected cornea. Methods Balb/c 3T3 fibroblasts were inoculated with human adenovirus (HAdV) serotypes 8, 19, or 37 and observed for cytopathic effect. Viral growth titers were performed, and apoptosis was measured by TUNEL assay. Viral and host cytokine gene expression was assessed by RT-PCR in cultured Balb/c 3T3 fibroblasts and in the corneas of virus-injected Balb/c mice. Western blot analysis was performed to detect cell signaling in the virus-infected cornea. Results Only HAdV37 induced cytopathic effect in mouse cells. Viral gene expression was limited, and viral replication was not detected. Apoptotic cell death in HAdV37-infected Balb/c cells was evident 48 and 72 hours postinfection (P < .01). MCP-1, IL-6, KC, and IP-10 mRNA levels were increased maximally by 8.4, 9.6, 10.5, and 20.0-fold, respectively, at 30 to 90 minutes after HAdV37 infection. Similar cytokine elevations were observed in the corneas of Balb/c mice 4 hours after stromal injection of HAdV37, when viral gene expression for the viral capsid protein IIIa was not detected. Western blot showed increased phosphorylation of ERK1/2 at 4 and 24 hours after corneal infection. Conclusions Despite limited viral gene expression, HAdV37 infection of Balb/c 3T3 fibroblasts results in increased proinflammatory gene expression. A similar pattern of cytokine expression in the corneas of HAdV37-infected Balb/c mice suggests the mouse adenoviral keratitis model may be useful for the study of early innate immune responses in the adenovirus-infected corneal stroma. PMID:17471351

  5. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  6. 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

    BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.
METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. β-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.
RESULTS—Intravenous or intraperitoneal injection of AdCMVβGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVβGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVβGal caused a higher β-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with

  7. Molecular replication

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1986-01-01

    The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

  8. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  9. Characterisation of the Equine adenovirus 2 genome.

    PubMed

    Giles, Carla; Vanniasinkam, Thiru; Barton, Mary; Mahony, Timothy J

    2015-09-30

    Equine adenovirus 2 (EAdV-2) is one of two serotypes of adenoviruses known to infect equines. Initial studies did not associate EAdV-2 infections with any specific clinical syndromes, although more recent evidence suggests that EAdV-2 may be associated with clinical and subclinical gastrointestinal infections of foals and adults respectively. In contrast, Equine adenovirus 1 is well recognised as a pathogen associated with upper respiratory tract infections of horses. In this study the complete genome sequence of EAdV-2 is reported. As expected, genes common to the adenoviruses were identified. Phylogenetic reconstructions using selected EAdV-2 genes confirmed the classification of this virus within the Mastadenovirus genus, and supported the hypothesis that EAdV-2 and EAdV-1 have evolved from separate lineages within the adenoviruses. One spliced open reading frame was identified that encoded for a polypeptide with high similarity to the pIX and E1b_55K adenovirus homologues and was designated pIX_E1b_55K. In addition to this fused version of E1b_55K, a separate E1b_55K encoding gene was also identified. These polypeptides do not appear to have evolved from a gene duplication event as the fused and unfused E1b_55K were most similar to E1b_55K homologues from the Atadenovirus and Mastadenovirus genera respectively. The results of this study suggest that EAdV-2 has an unusual evolutionary history that warrants further investigation. PMID:26220513

  10. 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

  11. Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

    PubMed Central

    Padi, Megha; Korkhin, Anna; James, Robert L.; Adelmant, Guillaume; Yoon, Rosa; Guo, Luxuan; Berrios, Christian; Zhang, Ying; Calderwood, Michael A.; Velmurgan, Soundarapandian; Cheng, Jingwei; Marto, Jarrod A.; Hill, David E.; Cusick, Michael E.; Vidal, Marc; Florens, Laurence; Washburn, Michael P.; Litovchick, Larisa; DeCaprio, James A.

    2012-01-01

    The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. PMID:23093934

  12. Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

    PubMed

    Fine, Debrah A; Rozenblatt-Rosen, Orit; Padi, Megha; Korkhin, Anna; James, Robert L; Adelmant, Guillaume; Yoon, Rosa; Guo, Luxuan; Berrios, Christian; Zhang, Ying; Calderwood, Michael A; Velmurgan, Soundarapandian; Cheng, Jingwei; Marto, Jarrod A; Hill, David E; Cusick, Michael E; Vidal, Marc; Florens, Laurence; Washburn, Michael P; Litovchick, Larisa; DeCaprio, James A

    2012-01-01

    The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. PMID:23093934

  13. Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo

    PubMed Central

    Tyler, MA; Ulasov, IV; Sonabend, AM; Nandi, S; Han, Y; Marler, S; Roth, J; Lesniak, MS

    2008-01-01

    Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone. PMID:19078993

  14. 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

  15. Robotic arm

    SciTech Connect

    Kwech, H.

    1989-04-18

    A robotic arm positionable within a nuclear vessel by access through a small diameter opening and having a mounting tube supported within the vessel and mounting a plurality of arm sections for movement lengthwise of the mounting tube as well as for movement out of a window provided in the wall of the mounting tube is disclosed. An end effector, such as a grinding head or welding element, at an operating end of the robotic arm, can be located and operated within the nuclear vessel through movement derived from six different axes of motion provided by mounting and drive connections between arm sections of the robotic arm. The movements are achieved by operation of remotely-controllable servo motors, all of which are mounted at a control end of the robotic arm to be outside the nuclear vessel. 23 figs.

  16. Robotic arm

    SciTech Connect

    Kwech, Horst

    1989-04-18

    A robotic arm positionable within a nuclear vessel by access through a small diameter opening and having a mounting tube supported within the vessel and mounting a plurality of arm sections for movement lengthwise of the mounting tube as well as for movement out of a window provided in the wall of the mounting tube. An end effector, such as a grinding head or welding element, at an operating end of the robotic arm, can be located and operated within the nuclear vessel through movement derived from six different axes of motion provided by mounting and drive connections between arm sections of the robotic arm. The movements are achieved by operation of remotely-controllable servo motors, all of which are mounted at a control end of the robotic arm to be outside the nuclear vessel.

  17. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    PubMed Central

    Gao, Peng

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  18. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice.

    PubMed

    Xie, Yinli; Gao, Peng; Li, Zhiyong

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  19. Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

    PubMed

    Yuan, Ziguo; Zhang, Xiuxiang; Zhang, Shoufeng; Liu, Ye; Gao, Shengyan; Zhang, Fei; Xu, Huijuan; Wang, Xiaohu; Hu, Rongliang

    2008-05-01

    Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. PMID:18249007

  20. Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis

    PubMed Central

    Chintakuntlawar, Ashish V.; Zhou, Xiaohong; Rajaiya, Jaya; Chodosh, James

    2010-01-01

    Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins. PMID:20419141

  1. Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene tissue fixative and formalin.

    PubMed

    Kap, Marcel; Arron, Georgina I; Loibner, M; Hausleitner, Anja; Siaulyte, Gintare; Zatloukal, Kurt; Murk, Jean-Luc; Riegman, Peter

    2013-08-01

    Formalin fixation is known to inactivate most viruses in a vaccine production context, but nothing is published about virus activity in tissues treated with alternative, non-crosslinking fixatives. We used a model assay based on cell culture to test formalin and PAXgene Tissue fixative for their virus-inactivating abilities. MDCK, A549, and MRC-5 cells were infected with Influenza A virus, Adenovirus, and Cytomegalovirus, respectively. When 75% of the cells showed a cytopathic effect (CPE), the cells were harvested and incubated for 15 min, or 1, 3, 6, or 24 hours, with PBS (positive control), 4% formalin, or PAXgene Tissue Fix. The cells were disrupted and the released virus was used to infect fresh MDCK, A549, and MRC-5 cells cultured on cover slips in 24-well plates. The viral cultures were monitored for CPE and by immunocytochemistry (ICC) to record viral replication and infectivity. Inactivation of Adenovirus by formalin occurred after 3 h, while Influenza A virus as well as Cytomegalovirus were inactivated by formalin after 15 min. All three virus strains were inactivated by PAXgene Tissue fixative after 15 min. We conclude that PAXgene Tissue fixative is at least as effective as formalin in inactivating infectivity of Influenza A virus, Adenovirus, and Cytomegalovirus. PMID:24845590

  2. Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

    PubMed Central

    Liikanen, Ilkka; Ahtiainen, Laura; Hirvinen, Mari LM; Bramante, Simona; Cerullo, Vincenzo; Nokisalmi, Petri; Hemminki, Otto; Diaconu, Iulia; Pesonen, Sari; Koski, Anniina; Kangasniemi, Lotta; Pesonen, Saila K; Oksanen, Minna; Laasonen, Leena; Partanen, Kaarina; Joensuu, Timo; Zhao, Fang; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum—a possible indicator of immune response—increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy. PMID:23546299

  3. 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

  4. Oncolytic adenovirus with temozolomide induces autophagy and antitumor immune responses in cancer patients.

    PubMed

    Liikanen, Ilkka; Ahtiainen, Laura; Hirvinen, Mari L M; Bramante, Simona; Cerullo, Vincenzo; Nokisalmi, Petri; Hemminki, Otto; Diaconu, Iulia; Pesonen, Sari; Koski, Anniina; Kangasniemi, Lotta; Pesonen, Saila K; Oksanen, Minna; Laasonen, Leena; Partanen, Kaarina; Joensuu, Timo; Zhao, Fang; Kanerva, Anna; Hemminki, Akseli

    2013-06-01

    Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy. PMID:23546299

  5. Structure and Uncoating of Immature Adenovirus

    SciTech Connect

    Perez-Berna, A.J.; Mangel, W.; Marabini, R.; Scheres, S. H. W., Menendez-Conejero, R.; Dmitriev, I. P.; Curiel, D. T.; Flint, S. J.; San Martin, C.

    2009-09-18

    Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

  6. Fluorescent antibody responses to adenoviruses in humans.

    PubMed

    Ariyawansa, J P; Tobin, J O

    1976-05-01

    Specific IgG, IgA, and IgM immunoglobulin antibody responses to adenovirus infections were studied by the indirect immunofluorescent technique in six pairs of human sera obtained during acute and convalescent phases of the illness. In addition, 70 single specimens of sera showing adenovirus IgG antibody from different age groups from birth to the 60th year of life were titrated for the same antibody to adenovirus types 1, 2, 3, 5, and 7, and 170 serum specimens from the same age groups were screened for specific immunoglobulin antibodies against types 1 and 5. Specific immunoglobulin antibodies lacked type specificity and in acute infections measured heterologous antibody response as well. On the other hand, IgG antibodies detected in single specimens of sera by immunofluorescence correlate with surveys of the isolation of virus from patients and neutralizing antibody studies by other workers. Fluorescent antibodies appeared in all three fractions of the immunoglobulins in acute adenovirus infections. Although this technique may be used in the diagnosis of adenovirus infections there is no advantage compared to complement-fixation testing. However, the use of sera absorbed with group antigen may have a more useful place in serological epidemiology than in diagnostic work. In five pairs of sera obtained during acute and convalescent phases of adenoviral illness and in 70 random single specimens from different age groups, "T" antibodies were detected only in the IgG fraction. The paired sera did not show a significant rise to indicate the usefulness of "T" antibody study in diagnosis. PMID:180061

  7. Fluorescent antibody responses to adenoviruses in humans.

    PubMed Central

    Ariyawansa, J P; Tobin, J O

    1976-01-01

    Specific IgG, IgA, and IgM immunoglobulin antibody responses to adenovirus infections were studied by the indirect immunofluorescent technique in six pairs of human sera obtained during acute and convalescent phases of the illness. In addition, 70 single specimens of sera showing adenovirus IgG antibody from different age groups from birth to the 60th year of life were titrated for the same antibody to adenovirus types 1, 2, 3, 5, and 7, and 170 serum specimens from the same age groups were screened for specific immunoglobulin antibodies against types 1 and 5. Specific immunoglobulin antibodies lacked type specificity and in acute infections measured heterologous antibody response as well. On the other hand, IgG antibodies detected in single specimens of sera by immunofluorescence correlate with surveys of the isolation of virus from patients and neutralizing antibody studies by other workers. Fluorescent antibodies appeared in all three fractions of the immunoglobulins in acute adenovirus infections. Although this technique may be used in the diagnosis of adenovirus infections there is no advantage compared to complement-fixation testing. However, the use of sera absorbed with group antigen may have a more useful place in serological epidemiology than in diagnostic work. In five pairs of sera obtained during acute and convalescent phases of adenoviral illness and in 70 random single specimens from different age groups, "T" antibodies were detected only in the IgG fraction. The paired sera did not show a significant rise to indicate the usefulness of "T" antibody study in diagnosis. PMID:180061

  8. 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

  9. 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

  10. Pharmacological inhibition of β3 integrin reduces the inflammatory toxicities caused by oncolytic adenovirus without compromising anticancer activity

    PubMed Central

    Browne, Ashley; Tookman, Laura A.; Ingemarsdotter, Carin K.; Bouwman, Russell D.; Pirlo, Katrina; Wang, Yaohe; McNeish, Iain A.; Lockley, Michelle

    2015-01-01

    Adenoviruses have been clinically tested as anti-cancer therapies but their utility has been severely limited by rapid, systemic cytokine release and consequent inflammatory toxicity. Here we describe a new approach to tackling these dangerous side effects. Using human ovarian cancer cell lines as well as malignant epithelial cells harvested from the ascites of women with ovarian cancer, we show that tumour cells do not produce cytokines in the first 24 hours following in vitro infection with the oncolytic adenovirus dl922-947. In contrast, dl922-947 does induce inflammatory cytokines at early time points following intraperitoneal (IP) delivery in mice with human ovarian cancer IP xenografts. In these animals, cytokines originate predominantly in murine tissues, especially in macrophage-rich organs such as the spleen. We use a non-replicating adenovirus to confirm that early cytokine production is independent of adenoviral replication. Using β3 integrin knockout mice injected intraperitoneally with dl922-947 and β3 null murine peritoneal macrophages we confirm a role for macrophage cell surface β3 integrin in this dl922-947-induced inflammation. We present new evidence that co-administration of a cyclic RGD-mimetic specific inhibitor of β3 integrin significantly attenuates the cytokine release and inflammatory hepatic toxicity induced by dl922-947 in an IP murine model of ovarian cancer. Importantly, we find no evidence that β3 inhibition compromises viral infectivity and oncolysis in vitro or anticancer efficacy in vivo. By enabling safe, systemic delivery of replicating adenoviruses, this novel approach could have a major impact on the future development of these effective anti-cancer agents. PMID:25977332

  11. Multiple proteins bind to VA RNA genes of adenovirus type 2.

    PubMed Central

    Van Dyke, M W; Roeder, R G

    1987-01-01

    Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection. Images PMID:3561405

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

    PubMed Central

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

    2010-01-01

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

  13. 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

  14. Verapamil Enhances the Antitumoral Efficacy of Oncolytic Adenoviruses

    PubMed Central

    Gros, Alena; Puig, Cristina; Guedan, Sonia; Rojas, Juan José; Alemany, Ramon; Cascallo, Manel

    2010-01-01

    The therapeutic potential of oncolytic adenoviruses is limited by the rate of adenovirus release. Based on the observation that several viruses induce cell death and progeny release by disrupting intracellular calcium homeostasis, we hypothesized that the alteration in intracellular calcium concentration induced by verapamil could improve the rate of virus release and spread, eventually enhancing the antitumoral activity of oncolytic adenoviruses. Our results indicate that verapamil substantially enhanced the release of adenovirus from a variety of cell types resulting in an improved cell-to-cell spread and cytotoxicity. Furthermore, the combination of the systemic administration of an oncolytic adenovirus (ICOVIR-5) with verapamil in vivo greatly improved its antitumoral activity in two different tumor xenograft models without affecting the selectivity of this virus. Overall, our findings indicate that verapamil provides a new, safe, and versatile way to improve the antitumoral potency of oncolytic adenoviruses in the clinical setting. PMID:20179683

  15. Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several studies have demonstrated that administration of type I, II, or III interferons (IFN) delivered using a replication defective human adenovirus 5 (Ad5) vector is effective to control Foot-and-Mouth Disease (FMD) in cattle and swine during experimental infections. However, high doses are requi...

  16. 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

  17. 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

  18. Mouse Adenovirus Type 1 Infection of Natural Killer Cell-Deficient Mice

    PubMed Central

    Welton, Amanda R.; Gralinski, Lisa E.; Spindler, Katherine R.

    2008-01-01

    Natural killer (NK) cells contribute to the initial nonspecific response to viral infection, and viruses exhibit a range of sensitivities to NK cells in vivo. We investigated the role of NK cells in infection of mice by mouse adenovirus type 1 (MAV-1) using antibody-mediated depletion and knockout mice. MAV-1 causes encephalomyelitis and replicates to highest levels in brains. NK cell-depleted mice infected with MAV-1 showed brain viral loads 8-20 days p.i. that were similar to wild-type control non-depleted mice. Mice genetically deficient for NK cells behaved similarly to wild-type control mice with respect to brain viral loads and survival. We conclude that NK cells are not required to control virus replication in the brains of MAV-1-infected mice. PMID:18155121

  19. [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

  20. SPRi-based adenovirus detection using a surrogate antibody method.

    PubMed

    Abadian, Pegah N; Yildirim, Nimet; Gu, April Z; Goluch, Edgar D

    2015-12-15

    Adenovirus infection, which is a waterborne viral disease, is one of the most prevelant causes of human morbidity in the world. Thus, methods for rapid detection of this infectious virus in the environment are urgently needed for public health protection. In this study, we developed a rapid, real-time, sensitive, and label-free SPRi-based biosensor for rapid, sensitive and highly selective detection of adenoviruses. The sensing protocol consists of mixing the sample containing adenovirus with a predetermined concentration of adenovirus antibody. The mixture was filtered to remove the free antibodies from the sample. A secondary antibody, which was specific to the adenovirus antibody, was immobilized onto the SPRi chip surface covalently and the filtrate was flowed over the sensor surface. When the free adenovirus antibodies bound to the surface-immobilized secondary antibodies, we observed this binding via changes in reflectivity. In this approach, a higher amount of adenoviruses resulted in fewer free adenovirus antibodies and thus smaller reflectivity changes. A dose-response curve was generated, and the linear detection range was determined to be from 10 PFU/mL to 5000 PFU/mL with an R(2) value greater than 0.9. The results also showed that the developed biosensing system had a high specificity towards adenovirus (less than 20% signal change when tested in a sample matrix containing rotavirus and lentivirus). PMID:26232675

  1. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

    SciTech Connect

    Tsukamotot, A.S.; Ponticelli, A.; Berk, A.J.; Gaynor, R.B.

    1986-07-01

    Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G/sub 0/-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo /sup 32/P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G/sub 0/-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

  2. Oncolytic Adenovirus Loaded with L-carnosine as Novel Strategy to Enhance the Antitumor Activity.

    PubMed

    Garofalo, Mariangela; Iovine, Barbara; Kuryk, Lukasz; Capasso, Cristian; Hirvinen, Mari; Vitale, Andrea; Yliperttula, Marjo; Bevilacqua, Maria Assunta; Cerullo, Vincenzo

    2016-04-01

    Oncolytic viruses are able to specifically replicate, infect, and kill only cancer cells. Their combination with chemotherapeutic drugs has shown promising results due to the synergistic action of virus and drugs; the combinatorial therapy is considered a potential clinically relevant approach for cancer. In this study, we optimized a strategy to absorb peptides on the viral capsid, based on electrostatic interaction, and used this strategy to deliver an active antitumor drug. We used L-carnosine, a naturally occurring histidine dipeptide with a significant antiproliferative activity. An ad hoc modified, positively charged L-carnosine was combined with the capsid of an oncolytic adenovirus to generate an electrostatic virus-carnosine complex. This complex showed enhanced antitumor efficacy in vitro and in vivo in different tumor models. In HCT-116 colorectal and A549 lung cancer cell lines, the complex showed higher transduction ratio and infectious titer compared with an uncoated oncolytic adenovirus. The in vivo efficacy of the complex was tested in lung and colon cancer xenograft models, showing a significant reduction in tumor growth. Importantly, we investigated the molecular mechanisms underlying the effects of complex on tumor growth reduction. We found that complex induces apoptosis in both cell lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in future studies also for delivery of other bioactive drugs. Mol Cancer Ther; 15(4); 651-60. ©2016 AACR. PMID:26861248

  3. Aerosol stability of bovine adenovirus type 3.

    PubMed Central

    Elazhary, M A; Derbyshire, J B

    1979-01-01

    The WBR-1 strain of bovine adenovirus type 3 was suspended in Eagle's medium or bovine nasal secretion and atomized into a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. Impinger samples of the aerosols were collected seven minutes, one, two and three hours postgeneration, and titrated for infectivity in embryonic bovine kidney cell cultures. Under certain conditions of temperature and relative humidity, the virus was more stable in aerosols of Eagle's medium than in nasal secretion. The bovine adenovirus was usually inactivated more rapidly at 30% relative humidity than at 90% relative humidity and during aging of the aerosols the virus was inactivated more rapidly at 32 degrees C than at 6 degrees C. PMID:226247

  4. Persistence and reactivation of human adenoviruses in the gastrointestinal tract.

    PubMed

    Kosulin, K; Geiger, E; Vécsei, A; Huber, W-D; Rauch, M; Brenner, E; Wrba, F; Hammer, K; Innerhofer, A; Pötschger, U; Lawitschka, A; Matthes-Leodolter, S; Fritsch, G; Lion, T

    2016-04-01

    Reactivation of persistent human adenoviruses (HAdVs) is associated with high morbidity and mortality in paediatric haematopoietic stem cell transplant (HSCT) recipients. Although invasive HAdV infections mainly arise from the gastrointestinal (GI) tract, the specific sites of HAdV persistence are not well characterised. We prospectively screened biopsies from 143 non-HSCT paediatric patients undergoing GI endoscopy and monitored serial stool specimens from 148 paediatric HSCT recipients for the presence of HAdV by real-time PCR. Persistence of HAdV in the GI tract was identified in 31% of children, with the highest prevalence in the terminal ileum. In situ hybridisation and immunohistochemistry identified HAdV persistence in lymphoid cells of the lamina propria, whereas biopsies from five transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI tract and reactivations post transplant indicated a correlation of intestinal HAdV shedding pre-transplant with high risk of invasive infection. HAdV persistence in the GI tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV persistence and reactivation, whereas the intestinal epithelium is the main site of viral proliferation preceding dissemination. The findings have important implications for assessing the risk of life-threatening invasive HAdV infections. PMID:26711435

  5. 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

  6. 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

  7. 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

  8. Replication of Tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Ishikawa, Masayuki

    2016-08-01

    Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems. PMID:27296148

  9. Bacteriophage replication modules.

    PubMed

    Weigel, Christoph; Seitz, Harald

    2006-05-01

    Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online). PMID:16594962

  10. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  11. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  12. Structure, function and dynamics in adenovirus maturation

    DOE PAGESBeta

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  13. 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).

  14. 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 ...

  15. 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

  16. Genetic organization, size, and complete sequence of early region 3 genes of human adenovirus type 41.

    PubMed Central

    Yeh, H Y; Pieniazek, N; Pieniazek, D; Luftig, R B

    1996-01-01

    The complete nucleotide and predicted amino acid sequences for open reading frames (ORFs) of the human adenovirus type 41 (Ad41) early region 3 (E3) gene have been determined. The sequence of the Ad41 E3 gene (map units 74 to 83.9) consists of 3,373 nucleotides and has one TATA box and two polyadenylation signals (AATAAA). Analysis of the nucleotide sequence reveals that the E3 gene can encode six ORFs, designated RL1 to RL6. These are all expressed at the mRNA level, as determined by reverse transcription-PCR analysis of AD41-infected cell RNA. When compared with known E3 sequences of most other human adenoviruses deposited in GenBank, the sequences of RL1 to RL3 were found to be unique to subgroup F adenoviruses (Ad40 and Ad41). They encode putative proteins of 173 amino acids (19.4 kDa) and 276 amino acids (31.6 kDa) in one reading frame as well as a 59- amino-acid (6.7 kDa) protein in an overlapping reading frame. RL4 encodes a 90-amino-acid protein (10.1 kDa) with 40% homology to the Ad2 E3 10.4-kDa protein, which induces degradation of the epidermal growth factor receptor and functions together with the Ad2 E3 14.5-kDa protein to protect mouse cell lines against lysis. RL5 encodes a protein of 107 amino acid residues (12.3 kDa) and is analogous to the Ad E3 14.5-kDa protein. RL6 codes for a protein of 122 amino acids (14.7 kDa) that is analogous to the Ad2 14.7-kDa protein, which functions to protect Ad-infected cells from tumor necrosis factor-induced cytolysis. This finding of three unique (RL1 to RL3) E3 gene ORFs may explain why subgroup F adenoviruses differ substantially from other human adenoviruses in their host range; i.e., they replicate predominantly in the host's gastrointestinal rather than respiratory tract. A recent phylogenetic study that compared subgroup F Ad40 DNA sequences with representatives of subgroups B (Ad3), C (Ad2), and E (Ad4) reached a similar conclusion about the uniqueness of RL1 and RL2. PMID:8642703

  17. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    PubMed Central

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

  18. Archaeal DNA replication.

    PubMed

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  19. Capturing and concentrating adenovirus using magnetic anionic nanobeads.

    PubMed

    Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

    2016-01-01

    We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

  20. Capturing and concentrating adenovirus using magnetic anionic nanobeads

    PubMed Central

    Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

    2016-01-01

    We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

  1. Enhanced inactivation of adenovirus under polychromatic UV lamps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The US EPA has stipulated that a UV fluence (dose) of 186 mJ cm-2 is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date publi...

  2. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  3. Biodistribution and Safety Assessment of Bladder Cancer Specific Recombinant Oncolytic Adenovirus in Subcutaneous Xenografts Tumor Model in Nude Mice

    PubMed Central

    Wang, Fang; Wang, Zhiping; Tian, Hongwei; Qi, Meijiao; Zhai, Zhenxing; Li, Shuwen; Li, Renju; Zhang, Hongjuan; Wang, Wenyun; Fu, Shenjun; Lu, Jianzhong; Rodriguez, Ronald; Guo, Yinglu; Zhou, Liqun

    2012-01-01

    Background The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. Materials and Method Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific Uroplakin II (UP II) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. Results General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5×108 pfu or higher dose (5×109 pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5×109 pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. Conclusions Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5×107 pfu and 5×108 pfu intratumorally injection in mice, without any discernable effects on general health

  4. Armoring CRAds with p21/Waf-1 shRNAs: the next generation of oncolytic adenoviruses

    PubMed Central

    Höti, N; Chowdhury, WH; Mustafa, S; Ribas, J; Castanares, M; Johnson, T; Liu, M; Lupold, SE; Rodriguez, R

    2011-01-01

    Conditionally replicating adenoviruses (CRAds) represent a promising modality for the treatment of neoplastic diseases, including Prostate Cancer. Selectively replicating viruses can be generated by placing a tissue or cancer-specific promoter upstream of one or more of the viral genes required for replication (for example, E1A, E1B). We have previously reported multiple cellular processes that can attenuate viral replication, which in turn compromises viral oncolysis and tumor kill. In this study, we investigated the importance of the cyclin-dependent kinase inhibitor p21/Waf-1, on viral replication and tumor growth. To our knowledge, this is the first report describing the importance of p21/Waf-1shRNA on the induction of an androgen responsive element (ARE) based promoter driving the E1A gene. As a proof of concept, the study emphasizes the use of RNA interference technology to overcome promoter weaknesses for tissue-specific oncolytic viruses, as well as the cellular inhibitor pathways on viral life cycle. Using RNA interference against p21/Waf-1, we were able to show an increase in viral replication and viral oncolysis of prostate cancer cells. Similarly, CRAd viruses that carry p21/Waf-1 shRNA (Ad5-RV004.21) were able to prevent tumor outgrowth that resulted in a marked increase in the mean survival time of tumor-bearing mice compared with CRAd without p21/Waf-1 shRNA (Ad5-RV004). In studies combining Ad5-RV004.21 with Adriamycin, a suprar-additive effect was observed only in CRAds that harbor shRNA against p21/Waf-1. Taken together, these findings of enhanced viral replication in prostate cancer cells by using RNA interference against the cdk inhibitor p21/Waf-1 have significant implications in the development of prostate-specific CRAd therapies. PMID:20448671

  5. Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge.

    PubMed

    Fischer, Laurent; Tronel, Jean Phillipe; Pardo-David, Camilla; Tanner, Patrick; Colombet, Guy; Minke, Jules; Audonnet, Jean-Christophe

    2002-10-01

    None of the currently available distemper vaccines provides a satisfactory solution for the immunization of very young carnivores in the face of maternal-derived immunity. Since mucosal immunization with replication-competent adenovirus-based vaccines has been proven effective in the face of passive immunity against the vector, it has the potential to provide a solution for the vaccination of young puppies born to canine distemper virus (CDV)-immune dams. We report the engineering and the characterization of two replication-competent canine adenovirus type 2 (CAV2)-based vaccines expressing, respectively, the CDV hemagglutinin (HA) and fusion (F) antigens. We first demonstrated that the intranasal vaccination with a mixture of both recombinant CAV2s provides an excellent level of protection in seronegative puppies, confirming the value of replication-competent adenovirus-based vectors for mucosal vaccination. In contrast, intranasal immunization with the same vaccine of puppies born to CDV- and CAV2-immune dams, failed to activate specific and protective immune responses. We hypothesized that an active CAV2 infection occurred while puppies were in close contact with the vaccinated dams in the breeding units and that the resulting active mucosal immunity interfered with the intranasal administration of CAV2-based CDV vaccine. However, when puppies born to CDV- and CAV2-immune dams were vaccinated subcutaneously with the CAV2-based CDV vaccine, significant seroconversion and solid protective immunity were triggered despite pre-existing systemic immunity to the vector. This latter result is surprising and suggests that subcutaneous vaccination with a replication-competent recombinant CAV2 may be an efficient strategy to overcome both passive and active adenovirus specific immunity in the dog. From a practical point of view, this could pave the way for an original strategy to vaccinate young puppies in the face of maternal-derived immunity. PMID:12297394

  6. E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

    PubMed

    Zheng, Yueting; Stamminger, Thomas; Hearing, Patrick

    2016-01-01

    Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection. PMID:26809031

  7. E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection

    PubMed Central

    Zheng, Yueting; Stamminger, Thomas; Hearing, Patrick

    2016-01-01

    Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection. PMID:26809031

  8. Arm CT scan

    MedlinePlus

    CAT scan - arm; Computed axial tomography scan - arm; Computed tomography scan - arm; CT scan - arm ... Mosby; 2013:chap 57. Shaw AS, Prokop M. Computed tomography. In: Adam A, Dixon AK, Gillard JH, Schaefer- ...

  9. Bicalutamide-activated oncolytic adenovirus for the adjuvant therapy of high-risk prostate cancer.

    PubMed

    Johnson, T J; Höti, N; Liu, C; Chowdhury, W H; Li, Y; Zhang, Y; Lupold, S E; Deweese, T; Rodriguez, R

    2013-07-01

    Conditionally replicating adenoviruses (CRAds) utilize tissue-specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer (PCa) gene therapy require androgens to activate prostate-specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen-suppressive therapy. We describe a novel prostate-specific CRAd generated by fusing the E1A gene to the androgen receptor (AR) cDNA with a point mutation in codon 685 (C685Y). The E1A-AR fusion neutralizes the previously described mutual inhibition of E1A and AR, and the C685Y point mutation alters specificity of steroid ligand binding to the AR, such that both androgens and nonsteroidal anti-androgens can activate viral replication. We demonstrate that the mutated E1A-AR retained the ability to function in regulating AR-responsive genes and E1A-responsive viral genes. In combination therapy of virus, bicalutamide (anti-androgen) and radiation, a profound impact on cell death by viral oncolysis was seen both in vitro and tumor xenografts. To our knowledge, this is the first gene therapy engineered to be enhanced by anti-androgens and a particularly attractive adjuvant strategy for intensity-modulated radiation therapy of high-risk PCas. PMID:23764901

  10. Bicalutamide Activated Oncolytic Adenovirus for the Adjuvant Therapy of High Risk Prostate Cancer

    PubMed Central

    Johnson, Tamara Jane; Hoti, Naser Uddin; Liu, Chunyan; Chowdhury, Wasim H.; Li, Ying; Zhang, Yonggang; Lupold, Shawn E.; DeWeese, Theodore; Rodriguez, Ronald

    2013-01-01

    Conditionally replicating adenoviruses (CRAds) utilize tissue specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer gene therapy require androgens to activate prostate specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen suppressive therapy. We describe a novel prostate specific CRAd generated by fusing the E1A gene to the androgen receptor (AR) cDNA with a point mutation in codon 685 (C685Y). The E1A-AR fusion neutralizes the previously described mutual inhibition of E1A & AR, and the C685Y point mutation alters specificity of steroid ligand binding to the AR, such that both androgens and non-steroidal anti-androgens can activate viral replication. We demonstrate that the mutated E1A-AR retained the ability to function in regulating AR responsive genes and E1A responsive viral genes. In combination therapy of virus, bicalutamide (anti-androgen) and radiation, a profound impact on cell death by viral oncolysis was seen both in vitro and tumor xenografts. To our knowledge, this is the first gene therapy engineered to be enhanced by anti-androgens, and a particularly attractive adjuvant strategy for intensity modulated radiation therapy (IMRT) of high-risk prostate cancers. PMID:23764901

  11. 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

  12. 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

  13. Biodistribution of an oncolytic adenovirus after intracranial injection in permissive animals: a comparative study of Syrian hamsters and cotton rats

    PubMed Central

    Sonabend, AM; Ulasov, IV; Han, Y; Rolle, CE; Nandi, S; Cao, D; Tyler, MA; Lesniak, MS

    2008-01-01

    Conditionally replicative adenoviruses (CRAds) are often evaluated in mice; however, normal and cancerous mouse tissues are poorly permissive for human CRAds. As the cotton rat (CR) is a semipermissive animal and the Syrian hamster (SH) is a fully permissive model for adenoviral replication, we compared them in a single study following intracranial (i.c.) injection of a novel glioma-targeting CRAd. Viral genomic copies were quantified by real-time PCR in brain, blood, liver and lung. The studies were corroborated by immunohistochemical, serological and immunological assays. CR had a multiple log higher susceptibility for adenoviral infection than SH. A similar amount of genomic copies of CRAd-Survivin-pk7 and human adenovirus serotype 5 (AdWT) was found in the brain of CR and in all organs from SH. In blood and lung of CR, AdWT had more genomic copies than CRAd-Survivin-pk7 in some of the time points studied. Viral antigens were confirmed in brain slices, an elevation of serum transaminases was observed in both models, and an increase in anti-adenoviral antibodies was detected in SH sera. In conclusion, CR represents a sensitive model for studying biodistribution of CRAds after i.c. delivery, allowing for the detection of differences in the replication of CRAd-Survivin-pk7 and AdWT that were not evident in SH. PMID:19011597

  14. 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

  15. 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

  16. 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.

  17. Deletion of the E4 region of the genome produces adenovirus DNA concatemers.

    PubMed Central

    Weiden, M D; Ginsberg, H S

    1994-01-01

    Two mutants containing large deletions in the E4 region of the adenovirus genome H5dl366 (91.9-98.3 map units) and H2dl808 (93.0-97.1 map units) were used to investigate the role of E4 genes in adenovirus DNA synthesis. Infection of KB human epidermoid carcinoma cells with either mutant resulted in production of large concatemers of viral DNA. Only monomer viral genome forms were produced, however, when mutants infected W162 cells, a monkey kidney cell line transformed with and expressing the E4 genes. Diffusible E4 gene products, therefore, complement the E4 mutant phenotype. The viral DNA concatemers produced in dl366- and dl808-infected KB cells did not have any specific orientation of monomer joining: the junctions consisted of head-to-head, head-to-tail, and tail-to-tail joints. The junctions were covalently linked molecules, but molecules were not precisely joined, and restriction enzyme maps revealed a heterogeneous size distribution of junction fragments. A series of mutants that disrupted single E4 open reading frames (ORFs) was also studied: none showed phenotypes similar to that of dl366 or dl808. Mutants containing defects in both ORF3 and ORF6, however, manifested the concatemer phenotype, indicating redundancy in genes preventing concatemer formation. These data suggest that the E4 ORFs 3 and 6 express functions critical for regulation of viral DNA replication and that concatemer intermediates may exist during adenovirus DNA synthesis. Images Fig. 2 Fig. 3 Fig. 4 PMID:8278357

  18. T-cell Subsets in Peripheral Blood and Tumors of Patients Treated With Oncolytic Adenoviruses

    PubMed Central

    Kristian, Taipale; Ilkka, Liikanen; Juuso, Juhila; Aila, Karioja-Kallio; Minna, Oksanen; Riku, Turkki; Nina, Linder; Johan, Lundin; Ari, Ristimäki; Anna, Kanerva; Anniina, Koski; Timo, Joensuu; Markus, Vähä-Koskela; Akseli, Hemminki

    2015-01-01

    The quality of the antitumor immune response is decisive when developing new immunotherapies for cancer. Oncolytic adenoviruses cause a potent immunogenic stimulus and arming them with costimulatory molecules reshapes the immune response further. We evaluated peripheral blood T-cell subsets of 50 patients with refractory solid tumors undergoing treatment with oncolytic adenovirus. These data were compared to changes in antiviral and antitumor T cells, treatment efficacy, overall survival, and T-cell subsets in pre- and post-treatment tumor biopsies. Treatment caused a significant (P < 0.0001) shift in T-cell subsets in blood, characterized by a proportional increase of CD8+ cells, and decrease of CD4+ cells. Concomitant treatment with cyclophosphamide and temozolomide resulted in less CD4+ decrease (P = 0.041) than cyclophosphamide only. Interestingly, we saw a correlation between T-cell changes in peripheral blood and the tumor site. This correlation was positive for CD8+ and inverse for CD4+ cells. These findings give insight to the interconnections between peripheral blood and tumor-infiltrating lymphocyte (TIL) populations regarding oncolytic virotherapy. In particular, our data suggest that induction of T-cell response is not sufficient for clinical response in the context of immunosuppressive tumors, and that peripheral blood T cells have a complicated and potentially misleading relationship with TILs. PMID:25655312

  19. The evolution of replicators.

    PubMed Central

    Szathmáry, E

    2000-01-01

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators. PMID:11127914

  20. 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.

  1. Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells ▿ †

    PubMed Central

    Puntener, Daniel; Engelke, Martin F.; Ruzsics, Zsolt; Strunze, Sten; Wilhelm, Corinne; Greber, Urs F.

    2011-01-01

    Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential. PMID:21047958

  2. Isolation and Epidemiology of Falcon Adenovirus

    PubMed Central

    Oaks, J. Lindsay; Schrenzel, Mark; Rideout, Bruce; Sandfort, Cal

    2005-01-01

    An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2005). In this study, the virus was successfully isolated and propagated in peregrine falcon embryo fibroblasts, in which it caused visible and reproducible cytopathology. Testing for serum neutralizing antibodies found that infection with this virus was limited almost exclusively to falcons. Serology also found that wild and captive peregrine falcons had high seropositivity rates of 80% and 100%, respectively, although clinical disease was rarely reported in this species. These data implicate peregrine falcons as the natural host and primary reservoir for the virus. Other species of North American falcons, including aplomado falcons, had lower seropositivity rates of 43 to 57%. Falcon species of tropical and/or island origin were uniformly seronegative, although deaths among adults of these species have been described, suggesting they are highly susceptible. Chickens and quail were uniformly seronegative and not susceptible to infection, indicating that fowl were not the source of infection. Based on the information from this study, the primary control of falcon adenovirus infections should be based on segregation of carrier and susceptible falcon species. PMID:16000467

  3. 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

  4. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2007-01-02

    Disclosed is a mutant CAR-DI-binding 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 a significantly weakened binding affinity for CAR-DI 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.

  5. Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01

    PubMed Central

    Vera, Beatriz; Martínez-Vélez, Naiara; Xipell, Enric; Acanda de la Rocha, Arlet; Patiño-García, Ana; Saez-Castresana, Javier; Gonzalez-Huarriz, Marisol; Cascallo, Manel; Alemany, Ramón; Alonso, Marta M.

    2016-01-01

    Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease. PMID:26808201

  6. Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01.

    PubMed

    Vera, Beatriz; Martínez-Vélez, Naiara; Xipell, Enric; Acanda de la Rocha, Arlet; Patiño-García, Ana; Saez-Castresana, Javier; Gonzalez-Huarriz, Marisol; Cascallo, Manel; Alemany, Ramón; Alonso, Marta M

    2016-01-01

    Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease. PMID:26808201

  7. Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro

    PubMed Central

    Lasek, Witold; Janyst, Michał; Młynarczyk, Grażyna

    2016-01-01

    There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells. PMID:26862302

  8. A simplified system for generating recombinant E3-deleted canine adenovirus-2.

    PubMed

    Yu, Zuo; Jiang, Qian; Liu, Jiasen; Guo, Dongchun; Quan, Chuansong; Li, Botao; Qu, Liandong

    2015-01-01

    Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector. PMID:25450764

  9. 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

  10. E1B and E4 Oncoproteins of Adenovirus Antagonize the Effect of Apoptosis Inducing Factor

    PubMed Central

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-01-01

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4orf3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4orf3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. PMID:24889240