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Sample records for activatable recombinant adenovirus

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

    PubMed

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

    2016-01-01

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

  7. [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

  8. Phylogenomic evidence for recombination of adenoviruses in wild gorillas.

    PubMed

    Hoppe, Eileen; Pauly, Maude; Robbins, Martha; Gray, Maryke; Kujirakwinja, Deo; Nishuli, Radar; Boji Mungu-Akonkwa, Dieu-Donné; Leendertz, Fabian H; Ehlers, Bernhard

    2015-10-01

    Human adenoviruses (HAdVs) of species Human mastadenovirus B (HAdV-B) are genetically highly diverse and comprise several pathogenic types. AdVs closely related to members of HAdV-B infect African great apes and the evolutionary origin of HAdV-B has recently been determined in ancient gorillas. Genetic evidence for intra- and inter-species recombination has been obtained for AdVs of humans and captive great apes, but evidence from wild great apes is lacking. In this study, potential HAdV-B members of wild Eastern gorillas were analysed for evidence of recombination. One near-complete genome was amplified from primary sample material and sequenced, and from another six individuals genome fragments were obtained. In phylogenomic analysis, their penton base, pVII-pVI, hexon and fiber genes were compared with those of all publicly available HAdV-B full-genome sequences of humans and captive great apes. Evidence for intra-species recombination between different HAdV-B members of wild gorillas as well as between HAdV-B members of chimpanzees and gorillas was obtained. Since zoonotic AdVs have been reported to cause respiratory outbreaks in both humans and monkeys, and humans in West and Central Africa frequently hunt and butcher primates thereby increasing the chance of zoonotic transmission, such HAdV-B recombinants might widen the pool of potential human pathogens. PMID:26219820

  9. 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...

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

    PubMed

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

    2013-08-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

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

    PubMed

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

    2015-04-30

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

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

    PubMed

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

    2011-09-01

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

  14. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-01-01

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene). PMID:26782515

  15. Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation.

    PubMed

    Yang, Xiaoshan; Xu, Rong; Lin, Yajun; Zhen, Yongzhan; Wei, Jie; Hu, Gang; Sun, Hongfan

    2016-01-01

    The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes, and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene, and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy. PMID:27530145

  16. Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation

    PubMed Central

    Yang, Xiaoshan; Xu, Rong; Lin, Yajun; Zhen, Yongzhan; Wei, Jie; Hu, Gang; Sun, Hongfan

    2016-01-01

    The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes, and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene, and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy. PMID:27530145

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

    PubMed

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

    2015-01-01

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

  18. Homologous Recombination in E3 Genes of Human Adenovirus Species D

    PubMed Central

    Singh, Gurdeep; Robinson, Christopher M.; Dehghan, Shoaleh; Jones, Morris S.; Dyer, David W.; Seto, Donald

    2013-01-01

    Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber. PMID:24027303

  19. 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

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

    SciTech Connect

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

    2014-02-15

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

  1. Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene.

    PubMed Central

    Wang, Y; Krushel, L A; Edelman, G M

    1996-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control

  2. Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

    SciTech Connect

    Li, Xiaohua; Fan, Rui; Zou, Xue; Gao, Lin; Jin, Haifeng; Du, Rui; Xia, Lin; Fan, Daiming . E-mail: fandaim@yahoo.com.cn

    2007-06-29

    Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.

  3. The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

    PubMed

    Obenauer-Kutner, Linda J; Ihnat, Peter M; Yang, Tong-Yuan; Dovey-Hartman, Barbara J; Balu, Arthi; Cullen, Constance; Bordens, Ronald W; Grace, Michael J

    2002-09-20

    A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples. PMID:12396622

  4. 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.

  5. [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

  6. 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

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

    PubMed Central

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

    2014-01-01

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

  8. 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

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

    PubMed

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

    2014-02-01

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

  10. 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

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

    PubMed Central

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

    1995-01-01

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

  12. 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

  13. 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

  14. 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

  15. Application of recombinant adenoviruses expressing glycoprotein or nucleoprotein of rabies virus to Korean raccoon dogs

    PubMed Central

    Choi, Jiyoung; Kim, Ha-Hyun; Jo, Hyun-Ye; Choi, Sung-Suk; Kim, Jong-Taek; Cho, In-Soo; Kim, Hee-Won

    2015-01-01

    Purpose A new rabies vaccine for animals, including raccoon dogs, in Korea is needed to eradicate rabies infection. In this study, we constructed two recombinant adenoviruses expressing the glycoprotein or nucleoprotein of the rabies virus (RABV). We then investigated the safety and immunogenicity of these strains in raccoon dogs, depending on inoculation route. Materials and Methods Recombinant adenoviruses expressing the glycoprotein (Ad-0910G) or nucleoprotein (Ad-0910N) of rabies were constructed in 293A cells using an adenoviral system. One-year-old raccoon dogs underwent intramuscular (IM) inoculation or oral administration of the recombinant Ad-0910G and Ad-0910N. Clinical symptoms were observed and virus-neutralizing antibodies (VNA) against RABV were measured at 0, 2, 4, and 6 weeks after the immunization. Raccoons were considered positive if VNA titers were ≥ 0.1 IU/mL. Results Raccoon dogs inoculated with the combined Ad-0910G and Ad-0910N virus via the IM route did not exhibit any clinical sign of rabies during the observation period. All raccoon dogs (n = 7) immunized IM had high VNA titers, ranging from 0.17 to 41.6 IU/mL at 2 weeks after inoculation, but 70% (7/10) of raccoon dogs administered viruses via the oral route responded by 6 weeks after administration against RABV. Conclusion Raccoon dogs inoculated with Ad-0910G and Ad-0910N viruses showed no adverse effects. Immunization with the combined Ad-0910G and Ad-0910N strains may play an important role in inducing VNA against RABV in raccoon dogs. PMID:26273578

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

    PubMed

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

    2009-03-01

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

  17. Mucosal vaccination with recombinant adenovirus encoding nucleoprotein provides potent protection against influenza virus infection.

    PubMed

    Kim, So-Hee; Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H; Song, Man Ki; Chang, Jun

    2013-01-01

    Influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. For this reason, development of universal vaccines targeting conserved viral components is needed. In this study, we generated recombinant adenovirus (rAd) vaccine encoding nucleoprotein (NP) of A/PR/8/34 influenza virus, designated rAd/NP. BALB/c mice were immunized intranasally or sublingually with rAd/NP vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. We found that intranasal immunization of rAd/NP elicited strong mucosal IgA responses as well as stronger CD8 T-cell responses toward immunodominant K(d)-restricted NP147-155 epitope than sublingual immunization. Importantly, only single intranasal but not sublingual immunization of rAd/NP provides potent protection against both homologous and heterologous influenza virus challenges. These results suggest that recombinant rAd/NP could be a universal vaccine candidate for mucosal administration against influenza virus. PMID:24086536

  18. Recombinant influenza virus carrying human adenovirus epitopes elicits protective immunity in mice.

    PubMed

    Yang, Penghui; Li, Tieling; Liu, Na; Gu, Hongjing; Han, Lina; Zhang, Peirui; Li, Zhiwei; Wang, Zhaohai; Zhang, Shaogeng; Wang, Xiliang

    2015-09-01

    Human adenoviruses (HAdVs) are known to cause a broad spectrum of diseases in pediatric and adult patients. As this time, there is no specific therapy for HAdV infection. This study used reverse genetics (RG) to successfully rescue a recombinant influenza virus, termed rFLU/HAdV, with the HAdV hexon protein antigenic epitope sequence inserted in the influenza non-structural (NS1) protein gene. rFLU/HAdV morphological characteristics were observed using electron microscopy. Furthermore, BALB/c mice immunized twice intranasally (i.n.) with 10(4) TCID50 or 10(5) TCID50 rFLU/HAdV showed robust humoral, mucosal, and cell-mediated immune responses in vivo. More importantly, these specific immune responses could protect against subsequent wild-type HAdV-3 (BJ809) or HAdV-7 (BJ1026) challenge, showing a significant reduction in viral load and a noticeable alleviation of histopathological changes in the challenged mouse lung in a dose-dependent manner. These findings highlighted that recombinant rFLU/HAdV warrants further investigation as a promising HAdV candidate vaccine and underscored that the immuno-protection should be confirmed in primate models. PMID:26112646

  19. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer With Recombinant Adenovirus in Nonhuman Primates

    PubMed Central

    Fontanellas, Antonio; Hervás-Stubbs, Sandra; Mauleón, Itsaso; Dubrot, Juan; Mancheño, Uxua; Collantes, María; Sampedro, Ana; Unzu, Carmen; Alfaro, Carlos; Palazón, Asis; Smerdou, Cristian; Benito, Alberto; Prieto, Jesús; Peñuelas, Iván; Melero, Ignacio

    2010-01-01

    Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector. PMID:20087317

  20. In vitro assessments of the genetic stability of a live recombinant human adenovirus vaccine against rabies.

    PubMed Central

    Lutze-Wallace, C; Sapp, T; Sidhu, M; Wandeler, A

    1995-01-01

    The genetic stability of a live human adenovirus 5: rabies glycoprotein recombinant vaccine has been assessed upon 20 serial passages in a permissive cell line of human origin. Restriction endonuclease analysis and the polymerase chain reaction were used to examine the integrity of the expression cassette for the rabies glycoprotein and the viral vector at the site of insertion of the cassette. It was found that the restriction endonuclease profile was identical for each sample assayed. A more detailed analysis of the expression cassette following amplification by the polymerase chain reaction revealed no changes in the size and number of fragments originating from the coding sequence for the glycoprotein nor the signals controlling the expression of the protein product. The amplified product obtained from the 10th and 20th passages was subjected to nucleotide sequencing. Additionally, 20 plaques isolated from the 20th passage of the virus expressed the rabies glycoprotein as demonstrated by fluorescent antibody staining with glycoprotein specific monoclonal antibodies. These results suggest that the recombinant vaccine maintains the integrity of the heterologous sequences upon passage in tissue culture. Images Fig. 1. Fig. 2. PMID:7648530

  1. Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene.

    PubMed

    Liu, Elizabeth B; Ferreyra, Leonardo; Fischer, Stephen L; Pavan, Jorge V; Nates, Silvia V; Hudson, Nolan Ryan; Tirado, Damaris; Dyer, David W; Chodosh, James; Seto, Donald; Jones, Morris S

    2011-01-01

    In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event. PMID:21915339

  2. In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

    PubMed Central

    Ballay, A; Levrero, M; Buendia, M A; Tiollais, P; Perricaudet, M

    1985-01-01

    We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor. Images Fig. 4. PMID:3004975

  3. Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

    PubMed Central

    Karayan, L; Hong, S S; Gay, B; Tournier, J; d'Angeac, A D; Boulanger, P

    1997-01-01

    Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle. PMID:9343226

  4. Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge.

    PubMed

    Hashem, Anwar; Jaentschke, Bozena; Gravel, Caroline; Tocchi, Monika; Doyle, Tracey; Rosu-Myles, Michael; He, Runtao; Li, Xuguang

    2012-04-01

    Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before. PMID:22370512

  5. [The induction of protective immune response in mice vaccinated by recombinant avian adenovirus CELO expressing glycoprotein G of the rabies virus].

    PubMed

    Shmarov, M M; Tutykhina, I L; Logunov, D Iu; Verkhovskai, L V; Nedosekov, V V; Tsybanov, S Zh; Novikov, B V; Narodnitskiĭ, B S; Gintsburg, A L

    2006-01-01

    The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive. PMID:16941876

  6. Approaches for genetic purity testing of live recombinant viral vaccines using a human adenovirus:rabies model.

    PubMed Central

    Lutze-Wallace, C; Sapp, T; Nadin-Davis, S A; Wandeler, A

    1992-01-01

    A two part purity testing regimen for genetically engineered live viral vaccines is described using a human adenovirus 5: rabies glycoprotein gene recombinant as a model vaccine. Initially, restriction endonuclease analysis of the recombinant viral genome verified the integrity of the recombinant construct and identified the vector genome. The second stage employed the polymerase chain reaction to facilitate a more detailed study of the target rabies glycoprotein cassette. The size of the target region was predicted from known nucleic acid sequence information and compared to that obtained after electrophoresis with molecular weight standards. Digestion of the polymerase chain reaction product with a second restriction endonuclease cleaved the target into a number of small fragments. Resolution of the fragments by gel electrophoresis allowed analysis of the target region alone, verifying its identity and integrity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1477804

  7. Combination of Recombinant Adenovirus-p53 with Radiochemotherapy in Unresectable Pancreatic Carcinoma

    PubMed Central

    Li, Jin-luan; Cai, Yong; Zhang, Shan-wen; Xiao, Shao-wen; Li, Xiao-fan; Duan, You-jia; Li, Yong-heng; Xu, Bo; Yan, Kun

    2011-01-01

    Objective To assess the safety and efficacy of the combination of recombinant adenovirus-p53 (rAd-p53) with radiochemotherapy for treating unresectable pancreatic carcinoma. Methods The eligible patients received concurrent rAd-p53 intratumoral injection and radiochemotherapy. Intratumoral injection of rAd-p53 was guided by B ultrasound. Radiochemotherapy consisted of intensity-modulated radiotherapy (IMRT) at two dose levels and intravenous gemcitabine (Gem). For radiotherapy, gross target volume (GTV) and clinical target volume (CTV) were 55-60 Gy and 45-55 Gy in 25-30 fractions, respectively. Concurrent intravenous gemcitabine was administered at 350 mg/m2, weekly, for 6 weeks. The primary end points included toxicity, clinical benefit response (CBR) and disease control rate (DCR). The secondary end points included progression-free survival (PFS) and overall survival (OS). Results Fifteen eligible patients were enrolled. Eight patients (53.3%) were evaluated as CBR and 12 (80%) achieved DCR. The median PFS and OS were 6.7 and 13.8 months, respectively. One-year PFS and OS were 40.0% and 51.1%, respectively. There were 8 (53.3%) patients reported grade 3 toxicities including neutropenia (6 patients, 40%), fever (1 patient, 6.7%) and fatigue (1 patient, 6.7%). There was no grade 4 toxicity reported. Conclusion Combination of rAd-p53 in unresectable pancreatic carcinoma showed encouraging efficacious benefit and was well tolerated. Long-term follow-up is needed to confirm the improvement of PFS and OS. PMID:23467436

  8. Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

    PubMed Central

    Gao, Dan-Dan; Fu, Jia; Qin, Bo; Huang, Wen-Xiang; Yang, Chun; Jia, Bei

    2016-01-01

    AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6 (Hyper-IL-6, HIL-6) and hepatocyte growth factor (HGF) (Ad-HGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF (Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure (ACLF). METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or Ad-HGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes. RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1 (HMGB1), endotoxin, tumour necrosis factor (TNF)-α and interferon-γ were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. Likewise, reduced hepatic damage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGF-HIL-6 treatment groups, more predominantly in the latter group. CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects. PMID:27122664

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

    PubMed

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

    2016-05-25

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

  10. Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.

    PubMed

    Müller, Claudia; Blenkinsop, Timothy A; Stern, Jeffrey H; Finnemann, Silvia C

    2016-01-01

    Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed β5-GFP after liposome-mediated transfection. The percentage of cells with detectable β5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing β5-GFP but increased variability of β5-GFP level among cells. In cells with low expression levels, β5-GFP localized mostly to the apical plasma membrane like endogenous αvβ5 integrin. In cells with high expression levels, β5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture. PMID:26427482

  11. 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

  12. Mucosal immunization with recombinant adenovirus encoding soluble globular head of hemagglutinin protects mice against lethal influenza virus infection.

    PubMed

    Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H; Song, Man Ki; Chang, Jun

    2013-12-01

    Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection against the virus infection. Combination of recombinant adenovirus (rAd) vector-based vaccine and mucosal administration is a promising regimen for safe and effective vaccination against influenza. In this study, we constructed rAd encoding the globular head region of HA from A/Puerto Rico/8/34 virus as vaccine candidate. The rAd vaccine was engineered to express high level of the protein in secreted form. Intranasal or sublingual immunization of mice with the rAd-based vaccine candidates induced significant levels of sustained HA-specific mucosal IgA and IgG. When challenged with lethal dose of homologous virus, the vaccinated mice were completely protected from the infection. The results demonstrate that intranasal or sublingual vaccination with HA-encoding rAd elicits protective immunity against infection with homologous influenza virus. This finding underlines the potential of our recombinant adenovirus-based influenza vaccine candidate for both efficacy and rapid production. PMID:24385946

  13. Cutting edge: recombinant adenoviruses induce CD8 T cell responses to an inserted protein whose expression is limited to nonimmune cells.

    PubMed

    Prasad, S A; Norbury, C C; Chen, W; Bennink, J R; Yewdell, J W

    2001-04-15

    CD8 T cells (T(CD8+)) play a crucial role in immunity to viruses. Current understanding of activation of naive T cells entails Ag presentation by professional APCs (pAPCs). What happens, however, when viruses evolve to avoid infecting pAPCs? We have studied the consequences of this strategy by generating recombinant adenoviruses that express influenza A virus nucleoprotein under the control of tissue-specific promoters. We show that the immunogenicity of such viruses requires their delivery to organs capable of expressing nucleoprotein. This indicates that infection of pAPCs is not required for adenoviruses to elicit a T(CD8+) response, probably due to a cross-priming via pAPCs. While this bodes well for recombinant adenoviruses as vaccines, it dims their prospects as gene therapy vectors. PMID:11290753

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

    PubMed

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

    2016-02-01

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

  15. In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

    PubMed

    Alyaqoub, Fadel S; Aldhamen, Yasser A; Koestler, Benjamin J; Bruger, Eric L; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Waters, Christopher M; Amalfitano, Andrea

    2016-02-15

    There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-β and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers. PMID:26792800

  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. Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children

    PubMed Central

    Wang, Yanqun; Li, Yamin; Lu, Roujian; Zhao, Yanjie; Xie, Zhengde; Shen, Jun; Tan, Wenjie

    2016-01-01

    Human adenoviruses (HAdVs) are prevalent in hospitalized children with severe acute respiratory infection (SARI). Here, we report a unique recombinant HAdV strain (CBJ113) isolated from a HAdV-positive child with SARI. The whole-genome sequence was determined using Sanger sequencing and high-throughput sequencing. A phylogenetic analysis of the complete genome indicated that the CBJ113 strain shares a common origin with HAdV-C2, HAdV-C6, HAdV-C1, HAdV-C5, and HAdV-C57 and formed a novel subclade on the same branch as other HAdV-C subtypes. BootScan and single nucleotide polymorphism analyses showed that the CBJ113 genome has an intra-subtype recombinant structure and comprises gene regions mainly originating from two circulating viral strains: HAdV-1 and HAdV-2. The parental penton base, pVI, and DBP genes of the recombinant strain clustered with the HAdV-1 prototype strain, and the E1B, hexon, fiber, and 100 K genes of the recombinant clustered within the HAdV-2 subtype, meanwhile the E4orf1 and DNA polymerase genes of the recombinant shared the greatest similarity with those of HAdV-5 and HAdV-6, respectively. All of these findings provide insight into our understanding of the dynamics of the complexity of the HAdV-C epidemic. More extensive studies should address the pathogenicity and clinical characteristics of the novel recombinant. PMID:26960434

  19. Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children.

    PubMed

    Wang, Yanqun; Li, Yamin; Lu, Roujian; Zhao, Yanjie; Xie, Zhengde; Shen, Jun; Tan, Wenjie

    2016-01-01

    Human adenoviruses (HAdVs) are prevalent in hospitalized children with severe acute respiratory infection (SARI). Here, we report a unique recombinant HAdV strain (CBJ113) isolated from a HAdV-positive child with SARI. The whole-genome sequence was determined using Sanger sequencing and high-throughput sequencing. A phylogenetic analysis of the complete genome indicated that the CBJ113 strain shares a common origin with HAdV-C2, HAdV-C6, HAdV-C1, HAdV-C5, and HAdV-C57 and formed a novel subclade on the same branch as other HAdV-C subtypes. BootScan and single nucleotide polymorphism analyses showed that the CBJ113 genome has an intra-subtype recombinant structure and comprises gene regions mainly originating from two circulating viral strains: HAdV-1 and HAdV-2. The parental penton base, pVI, and DBP genes of the recombinant strain clustered with the HAdV-1 prototype strain, and the E1B, hexon, fiber, and 100 K genes of the recombinant clustered within the HAdV-2 subtype, meanwhile the E4orf1 and DNA polymerase genes of the recombinant shared the greatest similarity with those of HAdV-5 and HAdV-6, respectively. All of these findings provide insight into our understanding of the dynamics of the complexity of the HAdV-C epidemic. More extensive studies should address the pathogenicity and clinical characteristics of the novel recombinant. PMID:26960434

  20. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.

    PubMed Central

    Yang, Y; Li, Q; Ertl, H C; Wilson, J M

    1995-01-01

    Recombinant adenoviruses are an attractive vehicle for gene therapy to the lung in the treatment of cystic fibrosis (CF). First-generation viruses deleted of E1a and E1b transduce genes into airway epithelial cells in vivo; however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. In this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive transfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. Our studies indicate that major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes are activated in response to newly synthesized antigens, leading to destruction of virus infected cells and loss of transgene expression. Major histocompatibility complex class II-associated presentation of exogenous viral antigens activates CD4+ T-helper (TH) cells of the TH1 subset and, to a lesser extent, of the TH2 subset. CD4+ cell-mediated responses are insufficient in the absence of cytotoxic T cells to completely eliminate transgene containing cells; however, they contribute to the formation of neutralizing antibodies in the airway which block subsequent adenovirus-mediated gene transfer. Definition of immunological barriers to gene therapy of cystic fibrosis should facilitate the design of rational strategies to overcome them. PMID:7884845

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Vaccination using recombinants influenza and adenoviruses encoding amastigote surface protein-2 are highly effective on protection against Trypanosoma cruzi infection.

    PubMed

    Barbosa, Rafael Polidoro Alves; Filho, Bruno Galvão; Dos Santos, Luara Isabela; Junior, Policarpo Ademar Sales; Marques, Pedro Elias; Pereira, Rafaela Vaz Sousa; Cara, Denise Carmona; Bruña-Romero, Oscar; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Machado, Alexandre Vieira

    2013-01-01

    In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease. PMID:23637908

  4. 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

  5. Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells

    PubMed Central

    2013-01-01

    Background Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects. Methods The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively. Results dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14ARF expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects. Conclusions Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the

  6. Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons

    PubMed Central

    Kim, Mi-La; Han, Shengjun; Lee, Sat-Byol; Kim, Jung Hye; Ahn, Hee Kyung

    2010-01-01

    Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression. PMID:21189997

  7. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  8. High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines.

    PubMed

    Wright, N; Jackson, F R; Niezgoda, M; Ellison, J A; Rupprecht, C E; Nel, L H

    2013-08-28

    Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine. PMID:23867013

  9. Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein.

    PubMed

    Hu, Chao; Zhu, Lihui; Luo, Rong; Dao, Jinwei; Zhao, Jiangping; Shi, Yaojun; Li, Hao; Lu, Ke; Feng, Xingang; Lin, Jiaojiao; Liu, Jinming; Cheng, Guofeng

    2014-11-01

    Schistosomiasis is a worldwide parasitic disease, and while it can be successfully treated with chemotherapy, this does not prevent reinfection with the parasite. Adenovirus vectors have been widely used for vaccine delivery, and a vaccination approach has the potential to prevent infection with Schistosoma. Here, we developed a recombinant adenoviral vector that expresses Schistosoma japonicum inhibitor apoptosis protein (Ad-SjIAP) and assessed its immunoprotective functions against schistosomiasis in mice. Murine immune responses following vaccination were investigated using enzyme-linked immunosorbent assays (ELISA), lymphocyte proliferation, and cytokine assays. The protective immunity in mice was evaluated by challenging with S. japonicum cercariae. Our results indicated that immunization with the Ad-SjIAP in mice induced a strong serum IgG response against IAP including IgG1, IgG2a, and IgG2b. In addition, lymphocyte proliferation experiments showed that mice treated with Ad-SjIAP significantly increased the lymphocyte response upon stimulation with recombinant Schistosoma japonicum inhibitor apoptosis protein (rSjIAP). Moreover, cytokine assays indicated that vaccination of Ad-SjIAP significantly increased the production of interferon (IFN)-γ and IL-2 as compared to the corresponding control group. Furthermore, following the challenge with S. japonicum cercariae, the vaccine conferred moderate protection, with an average rate of 37.95% for worm reduction and 31.7% for egg reduction. Taken together, our preliminarily results suggested that schistosoma IAP may be a potential vaccine against S. japonicum and that adenoviral vectors may serve as an alternative delivery vehicle for schistosome vaccine development. PMID:25185668

  10. Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria.

    PubMed

    Rodrigues, E G; Zavala, F; Eichinger, D; Wilson, J M; Tsuji, M

    1997-02-01

    The immunogenicity of a recombinant replication defective adenovirus expressing a major malaria Ag, the circumsporozoite (CS) protein (AdPyCS), was determined using a rodent malaria model. A single immunizing dose of this construct induced a large number of CS-specific CD8+ and CD4+ T cells in the spleens of these animals, particularly when given by the s.c. or i.m. route. A single dose of AdPyCS also induced high titers of Abs to Plasmodium yoelii sporozoites in mice. No other form of presentation of the CS protein given as a single immunizing dose, i.e., irradiated sporozoites, recombinant vaccinia, or influenza virus, etc., elicits comparably high numbers of CS-specific CD8+ T cells. The high concentration of CS-specific CD8+ T cells in the spleen was relatively short-lived, decreasing to half of its original value by 4 wk and to one-third at 8 wk after AdPyCS inoculation. The decrease in splenic CS-specific CD4+ T cells was even more rapid. Most importantly, a single dose of inoculation of AdPyCS into mice rendered them highly resistant to sporozoite challenge, resulting in a 93% inhibition of liver stage development of the parasites. This protective effect was primarily mediated by CD8+ T cells, as shown by depletion of this T cell population, while depletion of the CD4+ T cell population had only a minor effect on anti-plasmodial activity. Moreover, the inoculation of mice with AdPyCS induces sterile immunity in a significant proportion of mice, preventing the occurrence of parasitemia. PMID:9013969

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

    PubMed

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

    2000-08-01

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

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

    PubMed Central

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

    2000-01-01

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

  13. 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...

  14. Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

    PubMed Central

    Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A.; Marchevsky, Renato S.; Rocha, Maria Gabrielle L.; Dutra, Miriam S.; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T.

    2014-01-01

    Background Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Methodology/Principal Findings Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. Conclusions/Significance The

  15. Enhanced immune responses against Japanese encephalitis virus using recombinant adenoviruses coexpressing Japanese encephalitis virus envelope and porcine interleukin-6 proteins in mice.

    PubMed

    Liu, Hanyang; Wu, Rui; Liu, Kai; Yuan, Lei; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Yan, Qigui; Zhao, Qin; Wen, Xintian; Cao, Sanjie

    2016-08-15

    Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Previous studies have demonstrated that recombinant adenovirus serotype 5 (Ad5) may be a potential vaccine candidate because it can express JEV envelope epitopes and induce immune responses against JEV. Still, it will be necessary to develop an adjuvant that can enhance both humoral and cellular immune responses to the recombinant antigen delivered by non-replicating Ad5. In this study, we investigated the systemic immune responses of BALB/c mice immunized with recombinant adenovirus expressing JEV envelope epitopes in combination with porcine interleukin-6 (rAdE-IL-6).The rAdE-IL-6 immunized group had the highest titers of anti-JEV antibody as detected by an enzyme-linked immunosorbent assay (ELISA), as well as the highest levels of neutralizing antibody (1:75) as detected by a serum neutralization test. Similarly, higher concentrations of interferon-gamma (834.7pg/ml) and interleukin-6 (IL-6) (229.7pg/ml) were detected in the rAdE-IL-6 group using an ELISA assay. These data indicate that immunized BALB/c induce a strong cellular response against rAdE-IL-6. Furthermore, after challenge with the virulent JEV SCYA201201 strain, the rAdE-IL-6 group generated an immune protective response 70% greater than that of the control group, indicating that rAdE-IL-6 induced a protective immune response against JEV challenge in mice. The results from this study demonstrated that IL-6 is a strong adjuvant that can enhance both humoral and cellular immune responses in mice. Furthermore, a recombinant adenovirus coexpressing JEV envelope epitopes and porcine IL-6 protein may be an effective vaccine in animals. PMID:27235810

  16. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    PubMed Central

    Gahéry-Ségard, H; Molinier-Frenkel, V; Le Boulaire, C; Saulnier, P; Opolon, P; Lengagne, R; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A; Schatz, C; Courtney, M; Le Chevalier, T; Tursz, T; Guillet, J G; Farace, F

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein. PMID:9410899

  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. The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

    PubMed Central

    Gabitzsch, Elizabeth S.; Tsang, Kwong Yok; Palena, Claudia; David, Justin M.; Fantini, Massimo; Kwilas, Anna; Rice, Adrian E.; Latchman, Yvette; Hodge, James W.; Gulley, James L.; Madan, Ravi A.; Heery, Christopher R.; Balint, Joseph P.

    2015-01-01

    Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies. PMID:26374823

  20. TRAIL Recombinant Adenovirus Triggers Robust Apoptosis in Multidrug-Resistant HL-60/Vinc Cells Preferentially Through Death Receptor DR5

    PubMed Central

    Wu, Ching-Huang; Kao, Ching-Hai

    2008-01-01

    Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic because of its highly selective apoptosis-inducing action on neoplastic versus normal cells. However, some cancer cells express resistance to recombinant soluble TRAIL. To overcome this problem, we used a TRAIL adenovirus (Ad5/35-TRAIL) to induce apoptosis in a drug-sensitive and multidrug-resistant variant of HL-60 leukemia cells and determined the molecular mechanisms of Ad5/35-TRAIL-induced apoptosis. Ad5/35-TRAIL did not induce apoptosis in normal human lymphocytes, but caused massive apoptosis in acute myelocytic leukemia cells. It triggered more efficient apoptosis in drug-resistant HL-60/Vinc cells than in HL-60 cells. Treating the cells with anti-DR4 and anti-DR5 neutralizing antibodies (particularly anti-DR5) reduced, whereas anti-DcR1 antibody enhanced, the apoptosis triggered by Ad5/35-TRAIL. Whereas Ad5/35-TRAIL induced apoptosis in both cell lines through activation of caspase-3 and caspase-10, known to link the cell death receptor pathway to the mitochondrial pathway, it triggered increased mitochondrial membrane potential change (Δψm) only in HL-60/Vinc cells. Ad5/35-TRAIL also increased the production of reactive oxygen species, which play an important role in apoptosis. Therefore, using Ad5/35-TRAIL may be an effective therapeutic strategy for eliminating TRAIL-resistant malignant cells and these studies may provide clues to treat and eradicate acute myelocytic leukemias. PMID:18476767

  1. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB, an adenovirus-rabies recombinant vaccine.

    PubMed

    Brown, L J; Rosatte, R C; Fehlner-Gardiner, C; Bachmann, P; Ellison, J A; Jackson, F R; Taylor, J S; Davies, C; Donovan, D

    2014-02-12

    Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB, a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5 IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB. Serum RVNA of adults first tested positive (≥0.5 IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1-4 consecutive weeks; in adults the range was 2-15 weeks, with almost half of adults maintaining titres above 0.5 IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB, the best time to sample sera of wild adult foxes for evidence of vaccination is 7-11 weeks following bait distribution. Thirty-four foxes (25 ONRAB, 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination. PMID:24374501

  2. Targeted delivery of CYP2E1 recombinant adenovirus to malignant melanoma by bone marrow-derived mesenchymal stem cells as vehicles.

    PubMed

    Wang, Jishi; Ma, Dan; Li, Yan; Yang, Yuan; Hu, Xiaoyan; Zhang, Wei; Fang, Qin

    2014-03-01

    The aim of this study was to explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) as intermediate carriers on targeting of P450 gene recombinant adenovirus to malignant melanoma in vitro and in vivo. BMSCs were transduced with pAd5-CMV-CYP2E1 recombinant adenovirus. BMSC migration was detected by Transwell plates in vitro and by superparamagnetic iron oxide particles in vivo. Growth-inhibitory effect and apoptosis were determined by MTT and immunity fluorescence staining. Anticancer effects were examined by a human melanoma nude mouse model in vivo. BMSCs moved toward A375 cells in Transwell plates. Numerous superparamagnetic MSCs labeled with iron oxide were identified in the peripheral areas of the tumor, but were detected in primary organs by Prussian blue staining. BMSC-CYP2E1 cells mediated a bystander killing effect on CYP2E1-negative A375 cells during coculture (IC50 values for A375 cells cocultured with BMSC-EGFP and BMSC-CYP2E1 were 4.08 and 2.68 mmol/l, respectively). Intravenously injecting CYP2E1 recombinant adenovirus-loaded BMSCs in mice with established human melanoma managed to target the tumor site, and BMSCs with forced expression of CYP2E1 inhibited the growth of malignant cells in vivo by activating 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. BMSCs may serve as a platform of P450 gene-directed enzyme prodrug therapy for the delivery of chemotherapeutic prodrugs to tumors. PMID:24413391

  3. Activatable Molecular Probes for Cancer Imaging

    PubMed Central

    Lee, Seulki; Xie, Jin; Chen, Xiaoyuan

    2013-01-01

    The development of highly sensitive and specific molecular probes for cancer imaging still remains a daunting challenge. Recently, interdisciplinary research at the interface of imaging sciences and bionanoconjugation chemistry has generated novel activatable imaging probes that can provide high-resolution imaging with ultra-low background signals. Activatable imaging probes are designed to amplify output imaging signals in response to specific biomolecular recognition or environmental changes in real time. This review introduces and highlights the unique design strategies and applications of various activatable imaging probes in cancer imaging. PMID:20388112

  4. 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

  5. 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

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

    PubMed

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

    2002-11-01

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

  7. 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

  8. [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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Woodchuck dendritic cells generated from peripheral blood mononuclear cells and transduced with recombinant human adenovirus serotype 5 induce antigen-specific cellular immune responses.

    PubMed

    Ochoa-Callejero, Laura; Berraondo, Pedro; Crettaz, Julien; Olagüe, Cristina; Vales, Africa; Ruiz, Juan; Prieto, Jesús; Tennant, Bud C; Menne, Stephan; González-Aseguinolaza, Gloria

    2007-05-01

    Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines. PMID:17385694

  11. Thrombin-activatable fibrinolysis inhibitor.

    PubMed

    Marx, Pauline F

    2004-09-01

    The coagulation system is a potent mechanism that prevents blood loss after vascular injury. It consists of a number of linked enzymatic reactions resulting in thrombin generation. Thrombin converts soluble fibrinogen into a fibrin clot. The clot is subsequently removed by the fibrinolytic system upon wound healing. Thrombin-activatable fibrinolysis inhibitor (TAFI), which is identical to the previously identified proteins procarboxypeptidase B, R, and U, forms a link between blood coagulation and fibrinolysis. TAFI circulates as an inactive proenzyme in the bloodstream, and becomes activated during blood clotting. The active form, TAFIa, inhibits fibrinolysis by cleaving off C-terminal lysine residues from partially degraded fibrin that stimulates the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin. Consequently, removal of these lysines leads to less plasmin formation and subsequently to protection of the fibrin clot from break down. Moreover, TAFI may also play a role in other processes such as, inflammation and tissue repair. In this review, recent developments in TAFI research are discussed. PMID:15379716

  12. Response to Multiple Radiation Doses of Human Colorectal Carcinoma Cells Infected With Recombinant Adenovirus Containing Dominant-Negative Ku70 Fragment

    SciTech Connect

    Urano, Muneyasu; He Fuqiu; Minami, Akiko; Ling, C. Clifton; Li, Gloria C.

    2010-07-01

    Purpose: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. Methods and Materials: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. Results: Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was {approx}1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2-1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. Conclusion: Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

  13. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus

    PubMed Central

    de Cassan, Simone C.; Forbes, Emily K.; Douglas, Alexander D.; Milicic, Anita; Singh, Bijender; Gupta, Puneet; Chauhan, Virander S.; Chitnis, Chetan E.; Gilbert, Sarah C.; Hill, Adrian V. S.; Draper, Simon J.

    2011-01-01

    A central goal in vaccinology is the induction of high and sustained antibody responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent pre-clinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as alum to new pre-clinical adjuvants and adjuvants in clinical development such as Abisco®100, CoVaccine HT™, Montanide®ISA720 and SE-GLA, for their ability to induce high and sustained antibody responses and T cell responses. These adjuvants induced a broad range of antibody responses when used in a three-shot protein-in-adjuvant regime using the model antigen ovalbumin and leading blood-stage malaria vaccine candidate antigens. Surprisingly, this range of antibody immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost antibody responses primed by a human adenovirus serotype 5 (AdHu5) vaccine recombinant for the same antigen. This AdHu5-protein regime also induced a more cytophilic antibody response and demonstrated improved efficacy of merozoite surface protein-1 (MSP-1) protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination, and may circumvent the need for more potent chemical adjuvants. PMID:21813775

  14. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    SciTech Connect

    Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard . E-mail: bernhard.dietzschold@jefferson.edu

    2006-12-20

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.

  15. Immune responses in pigs induced by recombinant canine adenovirus 2 expressing the glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

    PubMed

    Zhou, J-X; Xue, J-D; Yu, T; Zhang, J-B; Liu, Y; Jiang, N; Li, Y-L; Hu, R-L

    2010-04-01

    To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection. PMID:20432066

  16. Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies.

    PubMed

    Hu, R L; Liu, Y; Zhang, S F; Zhang, F; Fooks, A R

    2007-07-20

    During the past decade, human rabies caused by cats has ranked the second highest in China. Several recombinant rabies vaccines have been developed for dogs. However, seldom have these vaccines been assessed or used in cats. In this trial, we report the experimental immunization of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3Delta-RGP, in cats. Thirty cats were inoculated with the recombinant vaccine intramuscularly, orally and intranasally, respectively. Safety and efficacy studies were undertaken using the fluorescent antibody virus neutralization (FAVN) test and evaluated. Results showed that this recombinant vaccine is safe for cats as demonstrated by the three different routes of administration. The vaccine stimulated an efficient humoral response in the vaccinated cats when 10(8.5)PFU/ml of the recombinant vaccine was injected intramuscularly in a single dose. The neutralizing antibody level increased above 0.5IU/ml at 4 weeks after the vaccination. The mean antibody level ranged from 0.96+/-0.26 to 4.47+/-1.57IU/ml among individuals, and the antibody levels were elicited for at least 12 months. After this period, the immunized cats survived the challenge of CVS-24 and an obvious anemnestic and protective immune response was stimulated after the challenge. The immune response occurred later than the inactivated vaccine and the overall antibody level in the vaccinated cats was lower, but it was sufficient to confer protection of cats against infection. This demonstrated that a single, intramuscular dose of CAV-2-E3Delta-RGP stimulated a long-lasting protective immune response in cats and suggested that CAV-2-E3Delta-RGP could be considered as a potential rabies vaccine candidate for cats. PMID:17576027

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

    PubMed

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

    2013-03-01

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

  18. Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR.

    PubMed

    Herbert, Rebecca; Baron, Jana; Batten, Carrie; Baron, Michael; Taylor, Geraldine

    2014-01-01

    Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine. PMID:24568545

  19. Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice.

    PubMed

    Wu, Yunpu; Qiao, Chuanling; Yang, Huanliang; Chen, Yan; Xin, Xiaoguang; Chen, Hualan

    2014-04-01

    The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 10(8) or 10(7). Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals. PMID:24688173

  20. Oral vaccination of dogs (Canis familiaris) with baits containing the recombinant rabies-canine adenovirus type-2 vaccine confers long-lasting immunity against rabies.

    PubMed

    Zhang, Shoufeng; Liu, Ye; Fooks, Anthony R; Zhang, Fei; Hu, Rongliang

    2008-01-17

    Rabies is a reemerging and fatal infectious disease in Asia mainly caused by exposure to rabid dogs. Prevention of dog rabies would be the most effective way to stop rabies transmission to humans. However, vaccinating stray dogs in urban and rural areas using conventional vaccines is always difficult and is not cost-effective for use in most areas including China. Further to previous studies from our laboratory, we developed a bait containing the recombinant rabies vaccine and performed a non-parenteral trial in dogs. This vaccine was intranasally administrated once to 46 dogs in solution form with 1 x 10(8.5) PFU and orally to 90 dogs in specially designed baits with 3 x 10(8.5) PFU of the recombinant canine adenovirus. Results showed that about 87.5% (119/136) of the immunized dogs developed virus neutralizing antibodies (VNA). The immune response against rabies in dogs was detectable at 2-3 weeks after administration, reaching a peak by 5-6 weeks. Among the seroconverted animals, 90.8% (108/119) elicited a VNA response for over 24 months. The antibody titer during the 2 years was above 0.5IU /ml while showing a gradual but slow decline from the 6th week after vaccination. In a challenge experiment of 10 dogs with 60,000 mouse LD(50) of CVS-24 2 years after the vaccination, all the dogs survived. This demonstrated that the recombinant vaccine could be orally administrated and the bait was effective for the oral vaccination of dogs. PMID:18083277

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

    PubMed

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

    2014-01-01

    The highly pathogenic avian influenza (HPAI) H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA) antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138), and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine. PMID:24671139

  2. Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung.

    PubMed

    Kaplan, J M; Armentano, D; Sparer, T E; Wynn, S G; Peterson, P A; Wadsworth, S C; Couture, K K; Pennington, S E; St George, J A; Gooding, L R; Smith, A E

    1997-01-01

    One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMV beta Gal vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the beta-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing beta-Gal from the E4+ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response. PMID:8989994

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

    PubMed Central

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

    2014-01-01

    The melanoma differentiation-associated gene-7 [MDA-7; renamed interleukin (IL)-24] was isolated from human melanoma cells induced to terminally differentiate by treatment with interferon and mezerein. MDA-7/IL-24 functions as a multimodality anticancer agent, possessing proapoptotic, antiangiogenic and immunostimulatory properties. All these attributes make MDA-7/IL-24 an ideal candidate for cancer gene therapy. In the present study, the human MDA-7/IL-24 gene was transfected into the human laryngeal cancer Hep-2 cell line and human umbilical vein endothelial cells (HUVECs) with a replication-incompetent adenovirus vector. Reverse transcription polymerase chain reaction and western blot analysis confirmed that the Ad-hIL-24 was expressed in the two cells. The expression of the antiapoptotic gene, Bcl-2, was significantly decreased and the IL-24 receptor was markedly expressed in Hep-2 cells following infection with Ad-hIL-24, but not in HUVECs. In addition, the expression of the proapoptotic gene, Bax, was induced and the expression of caspase-3 was increased in the Hep-2 cells and HUVECs. Methyl thiazolyl tetrazolium assay indicated that Ad-hIL-24 may induce growth suppression in Hep-2 cells but not in HUVECs. In conclusion, Ad-hIL-24 selectively inhibits proliferation and induces apoptosis in Hep-2 cells. No visible damage was found in HUVECs. Therefore, the results of the current study indicated that Ad-hIL-24 may have a potent suppressive effect on human laryngeal carcinoma cell lines, but is safe for healthy cells. PMID:24527085

  4. 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

  5. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    PubMed

    Gach, Johannes S; Gorlani, Andrea; Dotsey, Emmanuel Y; Becerra, Juan C; Anderson, Chase T M; Berzins, Baiba; Felgner, Philip L; Forthal, Donald N; Deeks, Steven G; Wilkin, Timothy J; Casazza, Joseph P; Koup, Richard A; Katlama, Christine; Autran, Brigitte; Murphy, Robert L; Achenbach, Chad J

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

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

    PubMed

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

    2011-01-01

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

  7. Medial hypothalamic 5-hydroxytryptamine (5-HT)1A receptors regulate neuroendocrine responses to stress and exploratory locomotor activity: application of recombinant adenovirus containing 5-HT1A sequences.

    PubMed

    Li, Qian; Holmes, Andrew; Ma, Li; Van de Kar, Louis D; Garcia, Francisca; Murphy, Dennis L

    2004-12-01

    Our previous studies found that serotonin transporter (SERT) knock-out mice showed increased sensitivity to minor stress and increased anxiety-like behavior but reduced locomotor activity. These mice also showed decreased density of 5-hydroxytryptamine (5-HT1A) receptors in the hypothalamus, amygdala, and dorsal raphe. To evaluate the contribution of hypothalamic 5-HT1A receptors to these phenotypes of SERT knock-out mice, two studies were conducted. Recombinant adenoviruses containing 5-HT1A sense and antisense sequences (Ad-1AP-sense and Ad-1AP-antisense) were used to manipulate 5-HT1A receptors in the hypothalamus. The expression of the 5-HT1A genes is controlled by the 5-HT1A promoter, so that they are only expressed in 5-HT1A receptor-containing cells. (1) Injection of Ad-1AP-sense into the hypothalamus of SERT knock-out mice restored 5-HT1A receptors in the medial hypothalamus; this effect was accompanied by elimination of the exaggerated adrenocorticotropin responses to a saline injection (minor stress) and reduced locomotor activity but not by a change in increased exploratory anxiety-like behavior. (2) To further confirm the observation in SERT-/- mice, Ad-1AP-antisense was injected into the hypothalamus of normal mice. The density and the function of 5-HT1A receptors in the medial hypothalamus were significantly reduced in Ad-1AP-antisense-treated mice. Compared with the control group (injected with Ad-track), Ad-1A-antisense-treated mice showed a significant reduction in locomotor activity, but again no changes in exploratory anxiety-like behaviors, tested by elevated plus-maze and open-field tests. Thus, the present results demonstrate that medial hypothalamic 5-HT1A receptors regulate stress responses and locomotor activity but may not regulate exploratory anxiety-like behaviors. PMID:15574737

  8. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects

    PubMed Central

    Gach, Johannes S.; Gorlani, Andrea; Dotsey, Emmanuel Y.; Becerra, Juan C.; Anderson, Chase T. M.; Berzins, Baiba; Felgner, Philip L.; Forthal, Donald N.; Deeks, Steven G.; Wilkin, Timothy J.; Casazza, Joseph P.; Koup, Richard A.; Katlama, Christine; Autran, Brigitte; Murphy, Robert L.; Achenbach, Chad J.

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  9. 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

  10. 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

  11. In vitro antitumor cytotoxic T lymphocyte response induced by dendritic cells transduced with DeltaNp73alpha recombinant adenovirus.

    PubMed

    Hu, Yijie; He, Yong; Srivenugopal, Kalkunte S; Fan, Shizhi; Jiang, Yaoguang

    2007-11-01

    DeltaNp73alpha, the N-terminal truncated form of p73alpha is a candidate tumor antigen because of its selective expression in many human cancers and lack of expression in normal tissues. Therefore, we investigated the effects of dendritic cells infected with adenoviral DeltaNp73alpha (DNp73alpha) on breaking immune tolerance and induction of immunity against DNp73alpha-expressing (A549 lung cancer, K-562 leukemia) and non-expressing (MCF-7 breast cancer) cell lines. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from a human umbilical cord blood were transduced with a recombinant adenoviral (Ad) vector encoding full-length human DNp73alpha cDNA (Ad-DNp73alpha) or a control vector Ad-EGFP, using the centrifugal force method. Induction of DNp73alpha-specific CTL response was evaluated by a cytotoxic assay against the three human tumor cell lines with different DNp73alpha expression levels. The viability and activation status of transduced dendritic cells were assessed by flow cytometry. The dendrocyte/Ad-DNp73alpha-activated cytotoxic T lymphocytes showed significantly higher cytotoxicity against the cell lines A549/DNp73alpha, K-562 that expressed DNp73alpha than the DNp73alpha-null MCF-7 cells. The DCs/Ad-DNp73alpha showed higher survival rates than the DCs/Ad-EGFP or untransduced DCs, presumably due to the inhibition of cell death. These findings, with potential applications for immunotherapy, demonstrate that dendrocytes transduced with Ad-DNp73alpha can induce specific and sustained T cell responses against tumors expressing this variant p53-related gene. PMID:17914557

  12. [Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

    PubMed

    Ma, Jing; Zhang, Xiao-Guang; Chen, Hong; Li, Kui-Biao; Zhang, Xiao-Mei; Zhang, Ke; Yang, Liang; Xu, Hong; Shu, Yue-Long; Tan, Wen-Jie; Zeng, Yi

    2009-09-01

    The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation. PMID:19954107

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

    PubMed Central

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

    1998-01-01

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

  14. Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination▿

    PubMed Central

    de Alencar, Bruna C. G.; Persechini, Pedro M.; Haolla, Filipe A.; de Oliveira, Gabriel; Silverio, Jaline C.; Lannes-Vieira, Joseli; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bruna-Romero, Oscar; Rodrigues, Mauricio M.

    2009-01-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  15. Perforin and gamma interferon expression are required for CD4+ and CD8+ T-cell-dependent protective immunity against a human parasite, Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination.

    PubMed

    de Alencar, Bruna C G; Persechini, Pedro M; Haolla, Filipe A; de Oliveira, Gabriel; Silverio, Jaline C; Lannes-Vieira, Joseli; Machado, Alexandre V; Gazzinelli, Ricardo T; Bruna-Romero, Oscar; Rodrigues, Mauricio M

    2009-10-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  16. Activatable Optical Probes for the Detection of Enzymes

    PubMed Central

    Drake, Christopher R.; Miller, David C.; Jones, Ella F.

    2013-01-01

    The early detection of many human diseases is crucial if they are to be treated successfully. Therefore, the development of imaging techniques that can facilitate early detection of disease is of high importance. Changes in the levels of enzyme expression are known to occur in many diseases, making their accurate detection at low concentrations an area of considerable active research. Activatable fluorescent probes show immense promise in this area. If properly designed they should exhibit no signal until they interact with their target enzyme, reducing the level of background fluorescence and potentially endowing them with greater sensitivity. The mechanisms of fluorescence changes in activatable probes vary. This review aims to survey the field of activatable probes, focusing on their mechanisms of action as well as illustrating some of the in vitro and in vivo settings in which they have been employed. PMID:23519774

  17. 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

  18. 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

  19. Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

    PubMed

    Hasegawa, K; Tagawa, M; Takagi, K; Tsukamoto, H; Tomioka, Y; Suzuki, T; Nishioka, Y; Ohrui, T; Numasaki, M

    2016-08-01

    Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy. PMID:27561689

  20. A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection

    PubMed Central

    2014-01-01

    As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4 × 109 infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0 × 1010 ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates. PMID:25363619

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  3. A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.

    PubMed

    Choi, Jin Huk; Jonsson-Schmunk, Kristina; Qiu, Xiangguo; Shedlock, Devon J; Strong, Jim; Xu, Jason X; Michie, Kelly L; Audet, Jonathan; Fernando, Lisa; Myers, Mark J; Weiner, David; Bajrovic, Irnela; Tran, Lilian Q; Wong, Gary; Bello, Alexander; Kobinger, Gary P; Schafer, Stephen C; Croyle, Maria A

    2015-08-01

    As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates. PMID:25363619

  4. Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs

    PubMed Central

    Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

    2015-01-01

    ABSTRACT Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against

  5. Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques

    SciTech Connect

    Schulte, Reiner; Suh, You-Suk; Sauermann, Ulrike; Ochieng, Washingtone; Sopper, Sieghart; Kim, Kwang S.; Ahn, So-Shin; Park, Ki S.; Stolte-Leeb, Nicole; Hunsmann, Gerhard; Sung, Young C. Stahl-Hennig, Christiane

    2009-01-20

    We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization. Both immunization strategies induced strong SIV Gag-specific IFN-{gamma} and T-cell proliferation responses and mediated a conservation of CD4{sup +} memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.

  6. 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

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

    PubMed Central

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

    2015-01-01

    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×109 IU/ml and 3.0×109 IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 109 IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ2MSB=20.00 and χ2WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development. PMID:25780403

  8. Goats Primed with Mycobacterium bovis BCG and Boosted with a Recombinant Adenovirus Expressing Ag85A Show Enhanced Protection against Tuberculosis

    PubMed Central

    Villarreal-Ramos, Bernardo; Nofrarías, Miquel; López-Soria, Sergio; Romera, Nadine; Singh, Mahavir; Abad, F. Xavier; Xing, Zhou; Vordermeier, H. Martin; Domingo, Mariano

    2012-01-01

    This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologous Mycobacterium bovis bacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 105 CFU of BCG (week 0), and 11 of them were boosted at week 8 with 109 PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 103 CFU of Mycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination and M. caprae infection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines. PMID:22761299

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

    PubMed Central

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

    1999-01-01

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

  10. 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

  11. [Disparity of apoptotic response in human breast cancer cell lines MCF-7 and MDA-MB-231 after infection with recombinant adenovirus encoding the VP2 gene of infectious bursail disease virus].

    PubMed

    Shin, Tan Seok; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd; Rahman, Sheikh-Omar Abdul

    2014-01-01

    Recombinant adenovirus encoding the VP2 gene of infectious bursal disease virus (ADV-VP2) has shown potent anti-tumour effects due to its capability of apoptotic induction in cancer cells. In the present study, human breast cancer cells MCF-7 and MDA-MB-231 were infected with ADV-VP2. The expression of VP2 protein was registered 4 h post-infection, particularly in MCF-7 cells. Multiple time-point DNA ladder assay demonstrated that ADV-VP2 infected MDA-MB-231 and MCF-7 cells endured apoptosis as early as 8 and 12 h post-infection, respectively. Apoptosis induction in both MDA-MB-231 and MCF-7 cells, albeit different start points, lasted til 36 h post-infection. The induction of apoptosis by ADV-VP2 was further shown by the TUNEL assay, with dark brown discoloration of apoptotic cells. The present study also explored the different stages of apoptosis by Annexin V/PI double staining flow cytometry quantification. Treated MCF-7 and MDA-MB-231 cells, respectively detected 25.58 +/- 9.02 and 14.51 +/- 3.12% of early apoptotic cells, 6.09 +/- 4.06 and 77.12 +/- 5.09% of late apoptotic cells. Results revealed that there were significant differences in the number of cells of both types which underwent early and late apoptosis. Significant differences were also observed among viable and apoptotic cells which have been post treated with ADV-VP2. The apoptotic effects of ADV-VP2 on human breast cancer cell lines were consistently demonstrated by three apoptosis detection methods. Therefore, a cancer vaccine basing on gene therapy could be developed in the near future using the present construct. PMID:25842834

  12. 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

  13. Photo-activatable Cre recombinase regulates gene expression in vivo.

    PubMed

    Schindler, Suzanne E; McCall, Jordan G; Yan, Ping; Hyrc, Krzystof L; Li, Mingjie; Tucker, Chandra L; Lee, Jin-Moo; Bruchas, Michael R; Diamond, Marc I

    2015-01-01

    Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods. PMID:26350769

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Extracellularly activatable nanocarriers for drug delivery to tumors

    PubMed Central

    Yeo, Yoon

    2014-01-01

    Introduction Nanoparticles for drug delivery to tumors need to satisfy two seemingly conflicting requirements: they should maintain physical and chemical stability during circulation and be able to interact with target cells and release drug at desired locations with no substantial delay. Unique microenvironment of tumors and externally-applied stimuli provide a useful means to maintain a balance between the two requirements. Areas covered We discuss nanoparticulate drug carriers that maintain stable structures in normal conditions but respond to stimuli for spatiotemporal control of drug delivery. We first define the desired effects of extracellular activation of nanoparticles and frequently used stimuli and review examples of extracellularly activated nanoparticles. Expert opinion Several challenges remain in developing extracellularly activatable nanoparticles. First, some of the stimuli-responsive NPs undergo incremental changes in response to stimuli, losing circulation stability. Second, the applicability of stimuli in clinical settings is limited due to the occasional occurrence of the activating conditions in normal tissues. Third, the construction of stimuli-responsive nanoparticles involves increasing complexity in nanoparticle structure and production methods. Future efforts are needed to identify new targeting conditions and increase the contrast between activated and non-activated NPs, while keeping the production methods simple and scalable. PMID:24950343

  19. Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

    PubMed

    Piotrowski, Christine; Ihling, Christian H; Sinz, Andrea

    2015-11-01

    Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner. PMID:25726908

  20. A two-photon activatable amino acid linker for the induction of fluorescence.

    PubMed

    Friedrich, Felix; Klehs, Kathrin; Fichte, Manuela A H; Junek, Stephan; Heilemann, Mike; Heckel, Alexander

    2015-10-28

    A new one- and two-photon activatable fluorophore based on ATTO565 was developed using a photolabile linker that simultaneously acts as a quencher. It is especially interesting for protein and peptide applications because it can be incorporated by standard peptide chemistry. The application of the new fluorogenic construct in super-resolution microscopy of antibody conjugates is shown. PMID:26343765

  1. An acid-cleavable phthalocyanine tetramer as an activatable photosensitiser for photodynamic therapy.

    PubMed

    Chow, Sun Y S; Lo, Pui-Chi; Ng, Dennis K P

    2016-08-16

    An acetal-linked self-quenched zinc(ii) phthalocyanine tetramer has been prepared. In an acidic environment in phosphate buffered saline or inside tumour cells, the phthalocyanine units of the tetramer are separated thereby restoring the fluorescence emission and singlet oxygen production. This response enables this compound to serve as a promising activatable photosensitiser for photodynamic therapy. PMID:27396392

  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. Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao

    2014-04-01

    pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence

  4. Activatable 19F MRI nanoparticle probes for the detection of reducing environments.

    PubMed

    Nakamura, Tatsuya; Matsushita, Hisashi; Sugihara, Fuminori; Yoshioka, Yoshichika; Mizukami, Shin; Kikuchi, Kazuya

    2015-01-12

    (19)F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the (19)F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction-responsive PFC-encapsulated nanoparticle probe, FLAME-SS-Gd(3+) (FSG). This is the first example of an activatable PFC-encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd(3+) complexes per nanoparticle was more than approximately 5.0×10(2), resulting in the high signal augmentation. PMID:25413833

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

    PubMed

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

    1990-08-01

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

  6. Extended Follow-up Confirms Early Vaccine-Enhanced Risk of HIV Acquisition and Demonstrates Waning Effect Over Time Among Participants in a Randomized Trial of Recombinant Adenovirus HIV Vaccine (Step Study)

    PubMed Central

    Duerr, Ann; Huang, Yunda; Buchbinder, Susan; Coombs, Robert W.; Sanchez, Jorge; del Rio, Carlos; Casapia, Martin; Santiago, Steven; Gilbert, Peter; Corey, Lawrence; Robertson, Michael N.

    2012-01-01

    Background. The Step Study tested whether an adenovirus serotype 5 (Ad5)–vectored human immunodeficiency virus (HIV) vaccine could prevent HIV acquisition and/or reduce viral load set-point after infection. At the first interim analysis, nonefficacy criteria were met. Vaccinations were halted; participants were unblinded. In post hoc analyses, more HIV infections occurred in vaccinees vs placebo recipients in men who had Ad5-neutralizing antibodies and/or were uncircumcised. Follow-up was extended to assess relative risk of HIV acquisition in vaccinees vs placebo recipients over time. Methods. We used Cox proportional hazard models for analyses of vaccine effect on HIV acquisition and vaccine effect modifiers, and nonparametric and semiparametric methods for analysis of constancy of relative risk over time. Results. One hundred seventy-two of 1836 men were infected. The adjusted vaccinees vs placebo recipients hazard ratio (HR) for all follow-up time was 1.40 (95% confidence interval [CI], 1.03–1.92; P = .03). Vaccine effect differed by baseline Ad5 or circumcision status during first 18 months, but neither was significant for all follow-up time. The HR among uncircumcised and/or Ad5-seropositive men waned with time since vaccination. No significant vaccine-associated risk was seen among circumcised, Ad5-negative men (HR, 0.97; P = 1.0) over all follow-up time. Conclusions. The vaccine-associated risk seen in interim analysis was confirmed but waned with time from vaccination. Clinical Trials Registration. NCT00095576. PMID:22561365

  7. Targeting the Genital Tract Mucosa with a Lipopeptide/Recombinant Adenovirus Prime/Boost Vaccine Induces Potent and Long-Lasting CD8+ T Cell Immunity Against Herpes: Importance of Myeloid Differentiation Factor 881

    PubMed Central

    Zhang, Xiuli; Dervillez, Xavier; Chentoufi, Aziz Alami; Badakhshan, Tina; Bettahi, Ilham; BenMohamed, Lbachir

    2012-01-01

    Targeting the mucosal immune system of the genital tract (GT) with subunit vaccines failed to induce potent and durable local CD8+ T cell immunity, crucial for protection against many sexually transmitted viral (STV) pathogens, including herpes simplex virus type 2 (HSV-2) that causes genital herpes. In this study, we aimed to investigate the potential of a novel lipopeptide/adenovirus type 5 (Lipo/rAdv5) prime/boost mucosal vaccine for induction of CD8+ T cell immunity to protect the female genital tract from herpes. The lipopeptide and the rAdv5 vaccine express the immunodominant HSV-2 CD8+ T cell epitope (gB498-505) and both were delivered intravaginally (IVAG) in the progesterone-induced B6 mouse model of genital herpes. Compared to its homologous lipopeptide/lipopeptide (Lipo/Lipo); the Lipo/rAdv5 prime/boost immunized mice: (i) developed potent and sustained HSV-specific CD8+ T cells, detected in both the GT draining nodes (GT-DLN) and in the vaginal mucosa (VM); (ii) had significantly lower virus titers; (iii) had decreased overt signs of genital herpes disease; and (iv) did not succumb to lethal infection (p < 0.005), following intravaginal HSV-2 challenge. Polyfunctional CD8+ T cells, producing IFN-γ, TNF-α and IL-2 and exhibiting cytotoxic activity, were associated with protection (p < 0.005). The protective CD8+ T cell response was significantly compromised in the absence of the adaptor myeloid differentiation factor 88 (MyD88) (p = 0.0001). Taken together, these findings indicate that targeting the VM with a Lipo/rAdv5 prime/boost vaccine elicits a potent, MyD88-dependent, and long-lasting mucosal CD8+ T cell protective immunity against sexually transmitted herpes infection and disease. PMID:23018456

  8. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  9. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

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

    PubMed Central

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

    2016-01-01

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

  11. Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

    PubMed Central

    Lin, Baochuan; Vora, Gary J.; Thach, Dzung; Walter, Elizabeth; Metzgar, David; Tibbetts, Clark; Stenger, David A.

    2004-01-01

    The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h. PMID:15243087

  12. 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

  13. 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

  14. H-type Dimer Formation of Fluorophores: A Mechanism for Activatable, in vivo Optical Molecular Imaging

    PubMed Central

    Ogawa, Mikako; Kosaka, Nobuyuki; Choyke, Peter L; Kobayashi, Hisataka

    2009-01-01

    In vivo molecular imaging with target-specific activatable “smart” probes, which only yield fluorescence at the intended target, enables sensitive and specific cancer detection because of high target to background ratios (TBR). Dimerization and fluorescence quenching has been shown to occur in concentrated aqueous solutions of various fluorophores. Here, we hypothesized that fluorophore dimerization and quenching after conjugation to targeting proteins can occur at low concentration, which is reasonable for in vivo imaging probes, because protein molecules can stabilize the fluorophore dimers based on physico-chemical interactions. This dimerization can be exploited as a mechanism for fluorescence activation. Rhodamine derivatives were conjugated to the cancer targeting molecules, avidin and trastuzumab, which target D-galactose receptor and HER2/neu antigen, respectively. After conjugation, a large proportion of R6G and TAMRA formed H-type dimers, even at low concentrations, but could be fully dequenched upon dissociation of the dimers to monomers. Lipophilicity was a potential factor in promoting H-dimer formation. To demonstrate the fluorescence activation effect during in vivo fluorescence endoscopic molecular imaging, a highly quenched probe, avidin-TAMRA or a minimally quenched probe, avidin-Alexa488 was administered into mice with ovarian metastases to the peritoneum. The tumors were clearly visualized with avidin-TAMRA, with low background fluorescence; in contrast, the background fluorescence was high for avidin-Alexa488. Thus, H-dimer formation as a mechanism of fluorescence quenching could be used to develop fluorescence activatable probes for in vivo molecular imaging. Effective activatable optical probes can be designed by focusing on the H-dimer formation of fluorophores. PMID:19480464

  15. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

    PubMed

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

    2016-09-10

    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis. PMID:27349354

  16. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

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

    2015-01-01

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

  17. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

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

    2015-01-01

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

  18. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao

    2012-03-01

    This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the

  19. 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

  20. One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

    PubMed

    Li, Li-Li; Wang, He-Rong; Zhou, Zhi-Yi; Luo, Jing; Xiao, Xiang-Qian; Wang, Xiao-Li; Li, Jin-Tao; Zhou, Yu-Bai; Zeng, Yi

    2016-04-01

    Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections. PMID:26821205

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    1998-03-01

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

  3. Recombinant influenza vaccines.

    PubMed

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  4. Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates

    PubMed Central

    Spring, Bryan Q.; Abu-Yousif, Adnan O.; Palanisami, Akilan; Rizvi, Imran; Zheng, Xiang; Mai, Zhiming; Anbil, Sriram; Sears, R. Bryan; Mensah, Lawrence B.; Goldschmidt, Ruth; Erdem, S. Sibel; Oliva, Esther; Hasan, Tayyaba

    2014-01-01

    Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease. PMID:24572574

  5. Activatable thermo-sensitive ICG encapsulated pluronic nanocapsules for temperature sensitive fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Kwong, Tiffany C.; Nouizi, Farouk; Sampathkumaran, Uma; Zhu, Yue; Alam, Maksudul M.; Gulsen, Gultekin

    2015-03-01

    Fluorescent tomography has been hindered by poor tissue penetration and weak signal which results in poor spatial resolution and quantification accuracy. Recently, it has been reported that activatable temperature responsive fluorescent probes which respond to focused ultrasound heating can improve the resolution and quantification of fluorescent tomography in deep tissue. This has lead to a new imaging modality, "Temperature-modulated fluorescent tomography." This technique relies on activatable thermo-sensitive fluorescent nanocapsules for whose fluorescence quantum efficiency is temperature dependent. Within a 4-5° C temperature range, the fluorescent signal increase more than 10-fold. In this molecular probe, Indocyanine Green (ICG) is encapsulated inside the core of a thermo-reversible pluronic micelle. Here we show the fluorescence response and temperature range of the nanocapsules which have been optimized for a higher temperature range to be used for in vivo animal imaging. We report on the feasibility of these temperature-sensitive reversible nanocapsules for in vivo applications by studying the pharmacokinetics in a subcutaneous mouse tumor model in vivo.

  6. Hydrogen peroxide-activatable antioxidant prodrug as a targeted therapeutic agent for ischemia-reperfusion injury

    PubMed Central

    Lee, Dongwon; Park, Seunggyu; Bae, Soochan; Jeong, Dahee; Park, Minhyung; Kang, Changsun; Yoo, Wooyoung; Samad, Mohammed A.; Ke, Qingen; Khang, Gilson; Kang, Peter M.

    2015-01-01

    Overproduction of hydrogen peroxide (H2O2) causes oxidative stress and is the main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury. Suppression of oxidative stress is therefore critical in the treatment of I/R injury. Here, we report H2O2-activatable antioxidant prodrug (BRAP) that is capable of specifically targeting the site of oxidative stress and exerting anti-inflammatory and anti-apoptotic activities. BRAP with a self-immolative boronic ester protecting group was designed to scavenge H2O2 and release HBA (p-hydroxybenzyl alcohol) with antioxidant and anti-inflammatory activities. BRAP exerted potent antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)- and H2O2-stimulated cells by suppressing the generation of ROS and pro-inflammatory cytokines. In mouse models of hepatic I/R and cardiac I/R, BRAP exerted potent antioxidant, anti-inflammatory and anti-apoptotic activities due to the synergistic effects of H2O2-scavenging boronic esters and therapeutic HBA. In addition, administration of high doses of BRAP daily for 7 days showed no renal or hepatic function abnormalities. Therefore BRAP has tremendous therapeutic potential as H2O2-activatable antioxidant prodrug for the treatment of I/R injuries. PMID:26563741

  7. Data supporting the structural and functional characterization of Thrombin‐Activatable Fibrinolysis Inhibitor in breast cancer

    PubMed Central

    Fawzy, Manal S.; Toraih, Eman A.

    2015-01-01

    The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015) [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC) progression (Zorio et al., 2008) [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005) [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors) will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer. PMID:26740968

  8. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    PubMed

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection. PMID:25962477

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

    PubMed Central

    Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

    2015-01-01

    Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

  10. 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

  11. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

    PubMed Central

    Henry, L J; Xia, D; Wilke, M E; Deisenhofer, J; Gerard, R D

    1994-01-01

    The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor. Images PMID:8035520

  12. Adenovirus-mediated expression of BmK CT suppresses growth and invasion of rat C6 glioma cells.

    PubMed

    Du, Jun; Fu, Yuejun; Wang, Jianing; Liang, Aihua

    2013-06-01

    BmK CT, one of the key toxins in the venom of the scorpion, Buthus martensii Karsch, can interact specifically with glioma cells as a chloride channel blocker and inhibit the invasion and migration of those cells via MMP-2. A recombinant adenovirus, Ad-BmK CT, was constructed and characterized by in vitro and in vivo studies, using MTT cytotoxicity assay and the glioma C6/RFP (red fluorescence protein)/BALB/c allogeneic athymic nude mice model, respectively. The adenovirus-mediated expression of BmK CT displayed a high activity in suppressing rat C6 glioma cells growth and invasion thereby suggesting that this recombinant adenovirus may be a powerful method for treating glioblastoma. PMID:23443213

  13. Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity.

    PubMed

    Machado, Alexandre V; Caetano, Bráulia C; Barbosa, Rafael P; Salgado, Ana Paula C; Rabelo, Renata H; Garcia, Cristiana C; Bruna-Romero, Oscar; Escriou, Nicolas; Gazzinelli, Ricardo T

    2010-04-19

    In this work, we explored an original vaccination protocol using recombinant influenza and adenovirus. We constructed recombinant influenza viruses harboring dicistronic NA segments containing the surface antigen 2 (SAG2) from Toxoplasma gondii under control of the duplicated 3' promoter. Recombinant influenza viruses were able to drive the expression of the foreign SAG2 sequence in cell culture and to replicate efficiently both in cell culture and in lungs of infected mice. In addition, mice primed with recombinant influenza virus and boosted with a recombinant adenovirus encoding SAG2 elicited both humoral and cellular immune responses specific for SAG2. Moreover, when immunized animals were challenged with the cystogenic P-Br strain of T. gondii, they displayed up to 85% of reduction in parasite burden. These results demonstrate the potential use of recombinant influenza vectors harboring the dicistronic segments in the development of vaccines against infectious diseases. PMID:20189485

  14. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob.

    PubMed

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-03-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 10(4) level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  15. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  16. Cucurbit[8]uril Regulated Activatable Supramolecular Photosensitizer for Targeted Cancer Imaging and Photodynamic Therapy.

    PubMed

    Wang, Xiao-Qiang; Lei, Qi; Zhu, Jing-Yi; Wang, Wen-Jing; Cheng, Qian; Gao, Fan; Sun, Yun-Xia; Zhang, Xian-Zheng

    2016-09-01

    Activatable photosensitizers (aPSs) have emerged as promising photodynamic therapy (PDT) agents for simultaneous imaging and selective ablation of cancer. However, traditional synthetic aPSs are limited by complex design and tedious synthesis. Here, aPS regulated by cucurbit[8]uril (CB[8]) for targeted cancer imaging and PDT is reported. This system is based on the host-guest interaction between biotinylated toluidine blue (TB-B) and CB[8] to form 2TB-B@CB[8]. Moreover, a facile strategy to turn off/on the fluorescence and photodynamic activity of TB-B is developed through the reversible assembly/disassembly of 2TB-B@CB[8]. This established system can achieve selective accumulation in tumor, light-up cancer imaging, and enhanced anticancer behavior. Therefore, this work provides a novel and promising strategy for the aPS build via simple and facile regulation of supramolecular chemistry. PMID:27513690

  17. Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm.

    PubMed

    Dubis, Joanna; Zuk, Natalia; Grendziak, Ryszard; Zapotoczny, Norbert; Pfanhauser, Monika; Witkiewicz, Wojciech

    2014-04-01

    Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 ± 10.1 vs. 54.2 ± 10.9%, P < 0.0001). TAFI activity positively correlated with the white blood cell count (r = 0.486, P < 0.005). The uPAR concentration in the AAA patients was statistically significantly higher than in the control group and positively correlated with TAFI activity (r = 0.409, P = 0.02). The levels of PAI-1 and D-dimers (fibrin fragments) were significantly higher in patients with AAA than in the control group (44.3 ± 17.5 vs. 21.7 ± 8.7 ng/ml and 1869.6 ± 1490.1 vs. 181.5 ± 188.6 ng/ml, respectively). Lowered activity of the fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded. PMID:24378973

  18. Sensitive and selective tumor imaging with novel and highly activatable fluorescence strategies

    NASA Astrophysics Data System (ADS)

    Urano, Yasuteru

    2008-02-01

    Nowadays, several tumor imaging modalities such as MRI, PET and fluorescence imaging techniques have been extensively investigated. One of the central problems associated with these conventional tumor-targeted imaging methods, however, is the fact that the signal contrast between tumor and surrounding tissues relies on the efficient targeting to the tumor and the rapid sequestration or excretion of unbound agent. Among these modalities, only fluorescence imaging technique has a significant feature, in that great signal activation could be achieved which potentially leads to the selective imaging of cancer with higher tumor-to-background ratio. In this symposium, I will present some examples of fluorescence cancer imaging based on highly activatable strategies with using precisely designed novel fluorescence probes. Recently, we developed highly sensitive fluorescence probes for β-galactosidase which is applicable for living cell system. By utilizing these probes, we could establish a novel and highly activatable strategy for sensitive and selective optical imaging of imbedded tumor in the peritoneum. We took a two step procedure in that a lectin is used to localize β-galactosidase to cancer cells as an activating enzyme, and subsequent administration of a highly-sensitive fluorescence probe for the enzyme have afforded remarkable fluorescence activation selectively in tumor mass. Since the tumor-targeted enzyme can catalyze numerous substrate turnovers, a great number of fluorescent molecules could be produced and hence the rapid and sensitive detection of tumor in vivo with high tumor-to-background ratio could be achieved. Moreover, the consequent close-up investigation using fluorescence microscopy revealed that cancer microfoci as small as 200 μm could be successfully visualized.

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

    PubMed

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

    2013-01-01

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

  20. 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

  1. 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.

  2. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  3. 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

  4. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice.

    PubMed

    Ragot, T; Vincent, N; Chafey, P; Vigne, E; Gilgenkrantz, H; Couton, D; Cartaud, J; Briand, P; Kaplan, J C; Perricaudet, M

    1993-02-18

    Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle. PMID:8437625

  5. 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

  6. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  7. 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.

  8. Chemical synthesis of a two-photon-activatable chemokine and photon-guided lymphocyte migration in vivo

    PubMed Central

    Chen, Xin; Tang, Shan; Zheng, Ji-Shen; Zhao, Ruozhu; Wang, Zhi-Peng; Shao, Wen; Chang, Hao-Nan; Cheng, Jing-Yuan; Zhao, Hui; Liu, Lei; Qi, Hai

    2015-01-01

    Chemokine-guided lymphocyte positioning in tissues is crucial for normal operation of the immune system. Direct, real-time manipulation and measurement of single-cell responses to chemokines is highly desired for investigating the cell biology of lymphocyte migration in vivo. Here we report the development of the first two-photon-activatable chemokine CCL5 through efficient one-pot total chemical synthesis in milligram scale. By spatiotemporally controlled photoactivation, we show at the single-cell level that T cells perceive the directional cue without relying on PI3K activities, which are nonetheless required for persistent migration over an extended period of time. By intravital imaging, we demonstrate artificial T-cell positioning in cutaneous tissues and lymph nodes. This work establishes a general strategy to develop high-quality photo-activatable protein agents through tailor-designed caging of multiple residues and highlights the potential of photo-activatable chemokines for understanding and potential therapeutic manipulation of cell positioning and position-controlled cell behaviours in vivo. PMID:26008852

  9. Fluorescence in vivo imaging of live tumor cells with pH-activatable targeted probes via receptor-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Asanuma, Daisuke; Urano, Yasuteru; Nagano, Tetsuo; Hama, Yukihiro; Koyama, Yoshinori; Kobayashi, Hisataka

    2009-02-01

    One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background. Here, we achieve specific in vivo tumor visualization with a newly-designed "activatable" targeted fluorescence probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody, Trastuzumab, or galactosyl serum albumin (GSA). As proof of concept, ex and in vivo imaging of two different tumor mouse models was performed: HER2-overexpressed lung metastasis tumor with Trastuzumab-pH probe conjugates and lectin-overexpressed i.p. disseminated tumor with GSA-pH probe conjugates. These pH-activatable targeted probes were highly specific for tumors with minimal background signal. Because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. Furthermore, this strategy was also applied to fluorescence endoscopy in tumor mouse models, resulting in specific visualization of tumors as small as submillimeter in size that could hardly detected by naked eyes because of their poor contrast against normal tissues. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.

  10. Nucleic acid hybridization for detection of cell culture-amplified adenovirus.

    PubMed Central

    Huang, C; Deibel, R

    1988-01-01

    A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Seven cloned probes, as well as total adenovirus type 2 (Ad2) and Ad16 genomic DNA, were tested by a nucleic acid hybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. Adenovirus DNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, total Ad2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 reference serotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-H required less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2) and pAd16-BD (containing the BamHI D fragment from Ad16) hybridized only with reference serotypes from the homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected 100% of all isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-F were 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAd16-BD were 100% (subgroup B) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included in this study for comparison. Images PMID:3230138

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

    DOEpatents

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  12. 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

  13. 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

  14. 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.

  15. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

    PubMed Central

    Fasbender, A; Lee, J H; Walters, R W; Moninger, T O; Zabner, J; Welsh, M J

    1998-01-01

    Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo. PMID:9649572

  16. Virulence Potential of Activatable Shiga Toxin 2d–Producing Escherichia coli Isolates from Fresh Produce

    PubMed Central

    Melton-Celsa, Angela R.; O'Brien, Alison D.; Feng, Peter C. H.

    2016-01-01

    Shiga toxin (Stx)–producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  17. Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

    PubMed

    Melton-Celsa, Angela R; O'Brien, Alison D; Feng, Peter C H

    2015-11-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  18. Activatable Ferritin Nanocomplex for Real-Time Monitoring of Caspase-3 Activation during Photodynamic Therapy.

    PubMed

    Wang, Jingjing; Zhang, Liwen; Chen, Minglong; Gao, Shi; Zhu, Lei

    2015-10-21

    One mechanism of photodynamic therapy (PDT) for the ablation of tumors is to induce apoptosis. Visualization of apoptosis during PDT in real-time is of great benefit for predicting and evaluating therapeutic outcomes. Herein, we engineered a highly stable and sensitive caspase-3 ferritin activatable probe (FABP/ZnPc) for simultaneous delivery of a photosensitizer (ZnPc) and real-time visualization of apoptosis during PDT. Upon near-infrared (NIR) light irradiation, ZnPc becomes active and initiates apoptosis, upon which the outer layer of the FABP/ZnPc is degraded by the apoptotic marker, caspase-3, to boost strong fluorescent signals, ultimately allowing real-time imaging of apoptosis. Our results demonstrate the utility of FABP/ZnPc as a tool for PDT and simultaneous imaging of caspase-3 activation in vitro and in vivo. Overall, the ability of FABP/ZnPc to image apoptosis during PDT will not only facilitate optimizing and personalizing the PDT strategy but is also important for understanding the mechanisms of PDT. PMID:26388178

  19. Nanoparticle-mediated delivery of a rapidly activatable prodrug of SN-38 for neuroblastoma therapy

    PubMed Central

    Alferiev, Ivan S.; Iyer, Radhika; Croucher, Jamie L.; Adamo, Richard F.; Zhang, Kehan; Mangino, Jennifer L.; Kolla, Venkatadri; Fishbein, Ilia; Brodeur, Garrett M.; Levy, Robert J.; Chorny, Michael

    2015-01-01

    Nanomedicine-based strategies have the potential to improve therapeutic performance of a wide range of anticancer agents. However, the successful implementation of nanoparticulate delivery systems requires the development of adequately sized nanocarriers delivering their therapeutic cargo to the target in a protected, pharmacologically active form. The present studies focused on a novel nanocarrier-based formulation strategy for SN-38, a topoisomerase I inhibitor with proven anticancer potential, whose clinical application is compromised by toxicity, poor stability and incompatibility with conventional delivery vehicles. SN-38 encapsulated in biodegradable sub-100 nm sized nanoparticles (NP) in the form of its rapidly activatable prodrug derivative with tocopherol succinate potently inhibited the growth of neuroblastoma cells in a dose- and exposure time-dependent manner, exhibiting a delayed response pattern distinct from that of free SN-38. In a xenograft model of neuroblastoma, prodrug-loaded NP caused rapid regression of established large tumors, significantly delayed tumor regrowth after treatment cessation and markedly extended animal survival. The NP formulation strategy enabled by a reversible chemical modification of the drug molecule offers a viable means for SN-38 delivery achieving sustained intratumoral drug levels and contributing to the potency and extended duration of antitumor activity, both prerequisites for effective treatment of neuroblastoma and other cancers. PMID:25770994

  20. CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery.

    PubMed

    Kheirolomoom, Azadeh; Ingham, Elizabeth S; Mahakian, Lisa M; Tam, Sarah M; Silvestrini, Matthew T; Tumbale, Spencer K; Foiret, Josquin; Hubbard, Neil E; Borowsky, Alexander D; Murphy, William J; Ferrara, Katherine W

    2015-12-28

    Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy. PMID:26471394

  1. Optimal dye-quencher pairs for the design of an "activatable" nanoprobe for optical imaging.

    PubMed

    Simard, Bryan; Tomanek, Boguslaw; van Veggel, Frank C J M; Abulrob, Abedelnasser

    2013-10-01

    Optical imaging offers high sensitivity and portability at low cost. The design of an optimal "activatable" imaging agent could greatly decrease the background noise and increase specificity of the signal. Five different molecules have been used to quench basal fluorescence of an enzyme substrate labeled with Cy5, Cy5.5 or IR800 at a distance of 8 amino acids (32 Å): a 6 nm gold nanoparticle (NP), a 20 nm and a 30 nm iron oxide (FeO) NP, the black hole quencher BHQ-3 and the IRdye quencher QC-1. The quenching efficiencies were 99% for QC1-IR800, 98% for QC1-Cy5.5, 96% for 30 nm FeO NP-Cy5.5, 89% for BHQ3-Cy5, 84% for BHQ3-Cy5.5, 77-90% for 6 nm gold NP-Cy5.5, depending on the number of dyes around the NP, 79% for 20 nm FeO NP-Cy5.5 and 77% for Cy5.5-Cy5. Signal activation upon cleavage by the matrix metalloproteinase MMP9 was proportional to the quenching efficiencies, ranging from 3-fold with Cy5.5-Cy5 to 67-fold with QC1-IR800. This independent work reports on the properties of the dyes and quenchers explaining the superior performance of QC-1 and 30 nm FeO NPs. PMID:23892541

  2. An Activatable Theranostic Nanomedicine Platform Based on Self-Quenchable Indocyanine Green-Encapsulated Polymeric Micelles.

    PubMed

    Liu, Lanxia; Ma, Guilei; Zhang, Chao; Wang, Hai; Sun, Hongfan; Wang, Chun; Song, Cunxian; Kong, Deling

    2016-06-01

    Self-quenchable indocyanine green (ICG)-encapsulated micelles with folic acid (FA)-targeting specificity (FA-ICG-micelles) were developed for biologically activatable photodynamic theranostics. FA-ICG-micelles were successfully prepared using the thin-film hydration method, which allows ICG to be encapsulated with a high drug loading that induces an efficient ICG-based quenched state. FA-ICG-micelles are initially in the "OFF" state with no fluorescence signal or phototoxicity, but they become highly fluorescent and phototoxic in cellular degradative environments. Importantly, via folate receptor-mediated endocytosis, the FA targeting of FA-ICG-micelles enhanced intracellular uptake and photodynamic therapy (PDT) efficacy. Systematic administration of FA-ICG-micelles to folate receptor-positive tumor-bearing mice elicited prolonged blood circulation, enhanced tumor accumulation and improved therapeutic efficiency compared to free ICG. Therefore, based on the FA-targeted specificity and switchable photoactivity, FA-ICG-micelles have potential for photodynamic theranostics in cancer. PMID:27319216

  3. Non-invasive manipulation of Drosophila behavior by two-photon excited red-activatable channelrhodopsin

    PubMed Central

    Hsiao, Po-Yen; Tsai, Chia-Lun; Chen, Ming-Chang; Lin, Yen-Yin; Yang, Shang-Da; Chiang, Ann-Shyn

    2015-01-01

    Scattering and absorption limit light penetration through inhomogeneous tissue. To reduce scattering, biochemists have shifted the wavelengths of excitation light for optogenetic actuators and fluorescent proteins to the orange-red range, while physicists have developed multiphoton technologies for deep tissue stimulation. We have built a rapid multiphoton spectroscopic screening system of genetically encoded red-activatable channelrhodopsin (ReaChR), and considered specific behaviors in transgenic Drosophila melanogaster as readouts to optimize the laser parameters for two-photon optogenetic activation. A wavelength-tunable optical parametric amplifier was adopted as the major light source for widefield two-photon excitation (TPE) of ReaChR. Our assays suggest that the optimized TPE wavelength of ReaChR is 1250 nm. Exploiting its capacity for optogenetic manipulation to induce macroscopic behavioral change, we realized rapid spectroscopic screening of genetically encoded effectors or indicators in vivo, and used modulation of ReaChR in the fly as a successful demonstration of such a system. PMID:26601000

  4. 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

  5. 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 ...

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2005-02-01

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

  8. 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

  9. Transition-Metal-Free CO-Releasing BODIPY Derivatives Activatable by Visible to NIR Light as Promising Bioactive Molecules.

    PubMed

    Palao, Eduardo; Slanina, Tomáš; Muchová, Lucie; Šolomek, Tomáš; Vítek, Libor; Klán, Petr

    2016-01-13

    Carbon monoxide-releasing molecules (CORMs) are chemical agents used to administer CO as an endogenous, biologically active molecule. A precise spatial and temporal control over the CO release is the major requirement for their applications. Here, we report the synthesis and properties of a new generation of transition-metal-free carbon monoxide-releasing molecules based on BODIPY chromophores (COR-BDPs) activatable by visible-to-NIR (up to 730 nm) light. We demonstrate their performance for both in vitro and in vivo experimental settings, and we propose the mechanism of the CO release based on steady-state and transient spectroscopy experiments and quantum chemical calculations. PMID:26697725

  10. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

    PubMed

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  11. Design and synthesis of phospholipase C and A2-activatable near-infrared fluorescent smart probes.

    PubMed

    Popov, Anatoliy V; Mawn, Theresa M; Kim, Soungkyoo; Zheng, Gang; Delikatny, E James

    2010-10-20

    The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A(1), A(2) (PLA(2)), C (PLC), and D (PLD) based on their site of action. Here, we report the design, synthesis, and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA(2) or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged black hole quencher-3 (BHQ-3) to the phospholipid headgroup or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C(6), C(12)) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids was assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC(6)-PyroC(6)-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA(2), Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA(2) was negligible due to steric hindrance at the sn-2 position. In contrast, the C(12)-spacered PyroC(12)-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA(2) and the best relative PLA(2)/PLC sensitivity, significantly outperforming previously known probes. These results open an avenue for future in vivo experiments and for new probes to detect PL activity. PMID:20882956

  12. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  13. 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

  14. Impurity of recombinant adeno-associated virus type 2 affects the transduction characteristics following subretinal injection in the rat.

    PubMed

    Shen, Wei-Yong; Lai, Yvonne K Y; Lai, Chooi-May; Rakoczy, P Elizabeth

    2004-02-01

    We recently reported that different purification methods of recombinant adeno-associated virus type 2 (rAAV2) affect the transduction characteristics following subretinal injection. In this study, we examined the roles of contaminant proteins from the HEK-293 cells and helper adenovirus, inactivation of helper adenovirus and cell stress induced by DNA-damaging agents in rAAV-mediated retinal transduction. Our results showed that contaminating factors/proteins resulting from the helper E1 deleted adenovirus are possibly responsible for efficient RPE transduction. Future studies of these factors will undoubtedly lead to development of new therapeutic approaches to PR- and RPE-specific retinal diseases. PMID:14659960

  15. 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

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

    PubMed Central

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

    1998-01-01

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

  17. XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic

    PubMed Central

    Haeckel, Akvile; Appler, Franziska; Ariza de Schellenberger, Angela; Schellenberger, Eyk

    2016-01-01

    Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli—a concept that could lead to efficient production of highly multifunctional drugs in the future. PMID:27295081

  18. Activatable albumin-photosensitizer nanoassemblies for triple-modal imaging and thermal-modulated photodynamic therapy of cancer.

    PubMed

    Hu, Dehong; Sheng, Zonghai; Gao, Guanhui; Siu, Fungming; Liu, Chengbo; Wan, Qian; Gong, Ping; Zheng, Hairong; Ma, Yifan; Cai, Lintao

    2016-07-01

    Photodynamic therapy (PDT) is a noninvasive and effective approach for cancer treatment. The main bottlenecks of clinical PDT are poor selectivity of photosensitizer and inadequate oxygen supply resulting in serious side effects and low therapeutic efficiency. Herein, a thermal-modulated reactive oxygen species (ROS) strategy using activatable human serum albumin-chlorin e6 nanoassemblies (HSA-Ce6 NAs) for promoting PDT against cancer is developed. Through intermolecular disulfide bond crosslinking and hydrophobic interaction, Ce6 photosensitizer is effectively loaded into the HSA NAs, and the obtained HSA-Ce6 NAs exhibit excellent reduction response, as well as enhanced tumor accumulation and retention. By the precision control of the overall body temperature instead of local tumor temperature increasing from 37 °C to 43 °C, the photosensitization reaction rate of HSA-Ce6 NAs increases 20%, and the oxygen saturation of tumor tissue raise 52%, significantly enhancing the generation of ROS for promoting PDT. Meanwhile, the intrinsic fluorescence and photoacoustic properties, and the chelating characteristic of porphyrin ring can endow the HSA-Ce6 NAs with fluorescence, photoacoustic and magnetic resonance triple-modal imaging functions. Upon irradiation of low-energy near-infrared laser, the tumors are completely suppressed without tumor recurrence and therapy-induced side effects. The robust thermal-modulated ROS strategy combined with albumin-based activatable nanophotosensitizer is highly potential for multi-modal imaging-guided PDT and clinical translation. PMID:27061266

  19. pH-Activatable MnO-Based Fluorescence and Magnetic Resonance Bimodal Nanoprobe for Cancer Imaging.

    PubMed

    Hsu, Benedict You Wei; Ng, Michael; Tan, Aaron; Connell, John; Roberts, Thomas; Lythgoe, Mark; Zhang, Yu; Wong, Siew Yee; Bhakoo, Kishore; Seifalian, Alexander M; Li, Xu; Wang, John

    2016-03-01

    Stimuli-responsive nanoprobes that combine both fluorescence and magnetic resonance imaging (MRI) are anticipated to be highly beneficial for tumor visualization with high imaging sensitivity. By employing an interfacial templating scheme, a pH-activatable fluorescence/MRI dual-modality imaging nanoprobe is successfully developed based on the coencapsulation of MnO nanoparticles and coumarin-545T inside a hybrid silica nanoshell. To promote cancer cell targeting with high-specificity, the nanoprobes are also conjugated with folic acid to establish a greater affinity for cancer cells that over-express folate receptors on their cell membrane. In the new nanosystem, MnO nanoparticles are shown to function as an efficient fluorescence quencher of coumarin-545T prior to cellular uptake. However, fluorescence recovery is achieved upon acidic dissolution of the MnO nanoparticles following receptor-mediated endocytosis into the low pH compartments of the cancer cells. Meanwhile, the Mn(2+) ions thus released are also shown to exert a strong T1 contrast enhancement in the cancer cells. Therefore, by demonstrating the dual-activatable MRI and fluorescence imaging in response to the low pH conditions, it is envisioned that these nanoprobes would have tremendous potential for emerging cancer-imaging modalities such as image-guided cancer therapy. PMID:26895111

  20. Self-Assembled Nanostructures Based on Activatable Red Fluorescent Dye for Site-Specific Protein Probing and Conformational Transition Detection.

    PubMed

    Yu, Yang; Huang, Yanyan; Hu, Fang; Jin, Yulong; Zhang, Guanxin; Zhang, Deqing; Zhao, Rui

    2016-06-21

    Smart and versatile nanostructures have demonstrated their effectiveness for biomolecule analysis and show great potential in digging insights into the structural/functional relationships. Herein, a nanoscale molecular self-assembly was constructed for probing the site-specific recognition and conformational changes of human serum albumin (HSA) with tunable size and emission. A tetraphenylethylene derivative TPE-red-COOH was used as the building block for tailoring fluorescence-silent nanoparticles. The highly specific and sensitive response to HSA was witnessed by the fast turn-on of the red fluorescence and simultaneous disassembly of the nanostructures, whereas various endogenous biomolecules cannot induce such response. The mechanism investigation indicates that the combination of multiple noncovalent interactions is the driving force for disassembling and trapping TPE-red-COOH into HSA. The resultant restriction of intramolecular rotation of TPE-red-COOH in the hydrophobic cavity of HSA induces the significant red emission. By using the fluorescence activatable nanosensor as the structural indicator, the stepwise conformational transitions of HSA during denaturing and the partial refolding of subdomain IIA of HSA were facilely visualized. Benefiting from its activatable signaling, sensitivity, and simplicity, such molecular assembly provides a kind of soft nanomaterial for site-specific biomolecule probing and conformational transition detection concerning their structure, function, and biomedical characteristics. PMID:27232658

  1. XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic.

    PubMed

    Haeckel, Akvile; Appler, Franziska; Ariza de Schellenberger, Angela; Schellenberger, Eyk

    2016-01-01

    Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli-a concept that could lead to efficient production of highly multifunctional drugs in the future. PMID:27295081

  2. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors

    PubMed Central

    Kim, Julius W.; Young, Jacob S.; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S.; Balyasnikova, Irina V.

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10−9 M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  3. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors.

    PubMed

    Kim, Julius W; Young, Jacob S; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S; Balyasnikova, Irina V

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  4. 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

  5. In vivo expression of adenovirus-mediated lacZ gene in murine nasal mucosa.

    PubMed

    Arimoto, Yukiko; Nagata, Hiroshi; Isegawa, Naohisa; Kumahara, Keiichiro; Isoyama, Kyoko; Konno, Akiyoshi; Shirasawa, Hiroshi

    2002-09-01

    Adenovirus is a good tool for transferring exogenous genes into various organs because the virus has a wide spectrum of infection. In this report, we demonstrate that a recombinant adenovirus, Ax1CAlacZ, can transfer an exogenous lacZ gene into murine nasal mucosa in vivo. The efficiency of the exogenous gene expression varied for different cell types and was improved by optimizing the method of administration. In the olfactory region, the olfactory epithelia, sustentacular cells and olfactory nerve efficiently expressed lacZ gene transferred by Ax1CAlacZ using either of two administration methods, dripping or injecting. In contrast, in the respiratory region, the respiratory epithelia but not the subepithelial tissues expressed lacZ gene transferred by Ax1CAlacZ, and the efficiency of the gene transfer, which was low when the virus was administered by nasal drops, was improved when the virus was administered by injection. Our study demonstrated that gene transfer mediated by adenovirus is more efficient in the olfactory epithelia than in the respiratory epithelia, and may be applicable to nasal or paranasal diseases such as olfactory epithelial disturbances. PMID:12403125

  6. 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

  7. Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

    NASA Astrophysics Data System (ADS)

    Han, Jianfeng; Zhao, Dong; Zhong, Zhirong; Zhang, Zhirong; Gong, Tao; Sun, Xun

    2010-03-01

    Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

  8. 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.

  9. 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

  10. 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

  11. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    PubMed

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. 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

  17. [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

  18. Polypyrrole-based nanotheranostics for activatable fluorescence imaging and chemo/photothermal dual therapy of triple-negative breast cancer

    NASA Astrophysics Data System (ADS)

    Park, Dongjin; Ahn, Kyung-Ohk; Jeong, Kyung-Chae; Choi, Yongdoo

    2016-05-01

    Here, we fabricated polypyrrole nanoparticles (PPys) (termed HA10-PPy, HA20-PPy, and HA40-PPy) doped with different average molecular weight hyaluronic acids (HAs) (10, 20, and 40 kDa, respectively), and evaluated the effect of molecular weight of doped HA on photothermal induction, fluorescence quenching, and drug loading efficiencies. Doxorubicin-loaded HA-doped PPys (DOX@HA-PPys) could be used for imaging and therapy of triple-negative breast cancer (TNBC). Fluorescence turn-on, stimuli-responsive drug release, and photo-induced heating of DOX@HA-PPys enabled not only activatable fluorescence imaging but also subsequent chemo/photothermal dual therapy for TNBC. In particular, we illustrated the potential usefulness of the photothermal effect of the nanoparticles for overcoming chemoresistance in TNBC.

  19. Polypyrrole-based nanotheranostics for activatable fluorescence imaging and chemo/photothermal dual therapy of triple-negative breast cancer.

    PubMed

    Park, Dongjin; Ahn, Kyung-Ohk; Jeong, Kyung-Chae; Choi, Yongdoo

    2016-05-01

    Here, we fabricated polypyrrole nanoparticles (PPys) (termed HA10-PPy, HA20-PPy, and HA40-PPy) doped with different average molecular weight hyaluronic acids (HAs) (10, 20, and 40 kDa, respectively), and evaluated the effect of molecular weight of doped HA on photothermal induction, fluorescence quenching, and drug loading efficiencies. Doxorubicin-loaded HA-doped PPys (DOX@HA-PPys) could be used for imaging and therapy of triple-negative breast cancer (TNBC). Fluorescence turn-on, stimuli-responsive drug release, and photo-induced heating of DOX@HA-PPys enabled not only activatable fluorescence imaging but also subsequent chemo/photothermal dual therapy for TNBC. In particular, we illustrated the potential usefulness of the photothermal effect of the nanoparticles for overcoming chemoresistance in TNBC. PMID:27004751

  20. Real-time In Vivo Molecular Detection of Primary Tumors and Metastases with Ratiometric Activatable Cell-Penetrating Peptides

    PubMed Central

    Savariar, Elamprakash N.; Felsen, Csilla N.; Nashi, Nadia; Jiang, Tao; Ellies, Lesley G.; Steinbach, Paul; Tsien, Roger Y.; Nguyen, Quyen T.

    2013-01-01

    Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 reemission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion. PMID:23188503

  1. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  2. 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

  3. Effect of reference spectra in spectral fitting to discriminate enzyme-activatable photoacoustic probe from intrinsic optical absorbers

    NASA Astrophysics Data System (ADS)

    Hirasawa, Takeshi; Okawa, Shinpei; Iwatate, Ryu J.; Kamiya, Mako; Urano, Yasuteru; Ishihara, Miya

    2016-03-01

    Multispectral photoacoustic (MS-PA) imaging has been researched to image molecular probes in the presence of strong background signals produced from intrinsic optical absorbers. Spectral fitting method (SFM) discriminates probe signals from background signals by fitting the PA spectra that are calculated from MS-PA images to reference spectra of the probe and background, respectively. Because hemoglobin is a dominant optical absorber in visible to near-infrared wavelength range, absorption spectra of hemoglobin have been widely used as reference background spectra. However, the spectra of background signals produced from heterogeneous biological tissue differ from the reference background spectra due to presence of other intrinsic optical absorbers and effect of optical scattering. Due to the difference, the background signals partly remain in the probe images. To image the probe injected in subcutaneous tumors of mice clearly, we added the melanosome absorption spectrum to the reference background spectra because skin contains nonnegligible concentration of melanosome and the spectrum is very similar to the scattering spectrum of biological tissue. The probe injected in the subcutaneous tumor of mice was an enzyme-activatable probe which show their original colors only in the presence of γ-glutamyltranspeptidase, an enzyme associated with cancer. The probes have been successfully used for rapid fluorescence imaging of cancer. As a result of MS-PA imaging, by considering the melanosome absorption spectrum, the background signals were successfully suppressed and then clearer probe image was obtained. Our MS-PA imaging method afforded successful imaging of tumors in mice injected with activatable PA probes.

  4. Near-Infrared Light-Activatable Microneedle System for Treating Superficial Tumors by Combination of Chemotherapy and Photothermal Therapy.

    PubMed

    Chen, Mei-Chin; Lin, Zhi-Wei; Ling, Ming-Hung

    2016-01-26

    Because of the aggressive and recurrent nature of cancers, repeated and multimodal treatments are often necessary. Traditional cancer therapies have a risk of serious toxicity and side effects. Hence, it is crucial to develop an alternative treatment modality that is minimally invasive, effectively treats cancers with low toxicity, and can be repeated as required. We developed a light-activatable microneedle (MN) system that can repeatedly and simultaneously provide photothermal therapy and chemotherapy to superficial tumors and exert synergistic anticancer effects. This system consists of embeddable polycaprolactone MNs containing a photosensitive nanomaterial (lanthanum hexaboride) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone supporting array patch. Because of this supporting array, the MNs can be completely inserted into the skin and embedded within the target tissue for locoregional cancer treatment. When exposed to near-infrared light, the embedded MN array uniformly heats the target tissue to induce a large thermal ablation area and then melts at 50 °C to release DOX in a broad area, thus destroying tumors. This light-activated heating and releasing behavior can be precisely controlled and switched on and off on demand for several cycles. We demonstrated that the MN-mediated synergistic therapy completely eradicated 4T1 tumors within 1 week after a single application of the MN and three cycles of laser treatment. No tumor recurrence and no significant body weight loss of mice were observed. Thus, the developed light-activatable MN with a unique embeddable feature offers an effective, user-friendly, and low-toxicity option for patients requiring long-term and multiple cancer treatments. PMID:26592739

  5. Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion

    PubMed Central

    Prost, Sandrine; Sheahan, Sharon; Rannie, Dominic; Harrison, David J.

    2001-01-01

    This study evaluates the utility of Cre-expressing adenovirus for deletion of floxed genes in primary cells using primary murine hepatocytes. Adenovirus infection was very efficient, even at very low MOI (>95% infection at a MOI of 6) and did not reduce viability. High level LacZ expression was cytotoxic to hepatocytes but Cre expression had no effect on viability. Cre-mediated recombination was completed within a timespan that permits experimentation during primary culture (>95% recombination after 24 h), independently of the number of floxed alleles per cell. Recombination did not induce p53 or produce cytological nuclear abnormalities (even in polyploid cells). Contrary to expectation, deletion of DNA ligase 1 did not alter cell cycle progression, although Cre expression hastens entry to S phase from G1, independently of the presence of floxed sequences. We conclude that adenovirus-mediated deletion of floxed alleles in primary cells is a straightforward and highly efficient tool for conducting preliminary studies of conditional gene targeting. Primary cells have advantages of differentiation, relative purity and ease of experimentation within controlled conditions, while avoiding confounding problems encountered in vivo (i.e. target cell specificity, kinetics and level of recombination, and elicitation of inflammatory and immune responses). This system could help identify important phenotypic effects and design and interpret in vivo studies. PMID:11504888

  6. 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

  7. 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.

  8. 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

  9. 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).

  10. Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

    PubMed

    Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

    2016-01-01

    Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage. PMID:26558992

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

    PubMed Central

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

    2014-01-01

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

  13. CD40 Ligand and GMCSF Coexpression Enhance the Immune Responses and Protective Efficacy of PCV2 Adenovirus Vaccine.

    PubMed

    Li, Delong; Huang, Yong; Du, Qian; Wang, Zhenyu; Chang, Lingling; Zhao, Xiaomin; Tong, Dewen

    2016-04-01

    Porcine circovirus 2 (PCV2) capsid protein (Cap) is the major structural protein that is responsible for neutralizing antibodies development and protective immunity, thus it is usually used to develop vaccines against porcine circovirus-associated disease (PCVAD). Porcine CD40 ligand (CD40L) and granulocyte-macrophage colony-stimulating factor (GMCSF) have positive immunostimulatory effects on immunocytes and have been applied in vaccine efficacy improvement as attractive adjuvant cytokines, respectively. However, whether these two cytokines can produce synergistic effect in vaccines still need to be further studied. In this study, porcine CD40L and GMCSF were inserted into recombinant adenoviruses to test the immunogenicity of PCV2 adenovirus vaccine in mice. Western blot and indirect immunofluorescence assay showed that Ad-Cap, Ad-CD40L-Cap, Ad-Cap-GMCSF, and Ad-CD40L-Cap-GMCSF were successfully constructed. Indirect ELISA and virus neutralizing assay showed that CD40L and GMCSF could enhance humoral immune responses, and PCV2 Cap-specific antibody titer and neutralizing activities were significantly higher in Ad-CD40L-Cap-GMCSF group than that in the other groups that just inserted either porcine CD40L or GMCSF in recombinant adenoviruses. Moreover, lymphocyte proliferation assay and cytokine release assay showed that CD40L and GMCSF enhanced the cellular immune responses of Ad-Cap, and had synergistic effects in lymphocyte proliferative activities and Th1-type cytokine production. Following PCV2 challenge, the viral loads in lungs of Ad-CD40L-Cap-GMCSF group were significantly lower compared with Ad-Cap, Ad-CD40L-Cap, and Ad-Cap-GMCSF group. Taken together, the results of this study demonstrated that CD40L and GMCSF could synergistically enhance the protective immune responses of PCV2 adenovirus vaccine, which would be used as a potent vaccine for the prevention and control of PCVAD. PMID:26982652

  14. Tracking novel adenovirus in environmental and human clinical samples: no evidence of endemic human adenovirus type 58 circulation in Córdoba city, Argentina.

    PubMed

    Ferreyra, L J; Giordano, M O; Martínez, L C; Barril, P A; Masachessi, G; Isa, M B; Poma, R; Rajal, V; Biganzoli, P; Nates, S V; Pavan, J V

    2015-05-01

    In recent years, several types of human adenovirus (HAdV) have arisen from the recombination between two or more previously known HAdV types, but their epidemiology is poorly understood. In this study, we investigated the circulation of HAdV-58, a recently described HAdV isolated from an HIV-positive patient in Córdoba city, Argentina. For this purpose, a 30-month survey was conducted to study the presence of this type of adenovirus in sewage samples collected at the inlet from a wastewater treatment plant in Córdoba city, Argentina. Complementarily, the virus was sought in stools of HIV-positive patients. Although HAdVs were detected in human stool samples and in a high percentage of sewage samples, no evidence of HAdV-58 circulation was detected. We suggest that there is no endemic circulation of HAdV-58 in the geographical local area. The trend is that the number of identified HAdVs increases over time. In this context, understanding the current circulating HAdVs may be biologically relevant. PMID:25165987

  15. 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...

  16. 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

  17. 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

  18. Photoacoustic Imaging: Semiconducting Oligomer Nanoparticles as an Activatable Photoacoustic Probe with Amplified Brightness for In Vivo Imaging of pH (Adv. Mater. 19/2016).

    PubMed

    Miao, Qingqing; Lyu, Yan; Ding, Dan; Pu, Kanyi

    2016-05-01

    Despite the great potential of photoacoustic imaging in the life sciences, the development of smart activatable photoacoustic probes remains elusive. On page 3662, K. Pu and co-workers report a facile nanoengineering approach based on semiconducting oligomer nano-particles to develop ratiometric photoacoustic probes with amplified brightness and enhanced sensing capability for accurate photoacoustic mapping of pH in the tumors of living mice. PMID:27167028

  19. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  20. Imaging Dendrimer-Grafted Graphene Oxide Mediated Anti-miR-21 Delivery With an Activatable Luciferase Reporter.

    PubMed

    Wang, Fu; Zhang, Beilei; Zhou, Lin; Shi, Yaru; Li, Zhiqiang; Xia, Yuqiong; Tian, Jie

    2016-04-13

    MicroRNAs (miRNAs) are a class of post-transcriptional gene regulators involved in various physiological processes including carcinogenesis, and they have emerged as potential targets for tumor theranostics. However, the employment of antisense oligonucleotides, termed anti-miRs, for antagonizing miRNA functions in vivo has largely been impeded by a lack of effective delivery carriers. Here, we describe the development of polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG)-functionalized nanographene oxide (NGO) conjugate (NGO-PEG-dendrimer) for the efficient delivery of anti-miR-21 into non-small-cell lung cancer cells. To monitor the delivery of anti-miR-21 into cells and tumors, we also constructed an activatable luciferase reporter (Fluc-3xPS) containing three perfectly complementary sequences against miR-21 in the 3' untranslated region (UTR) of the reporter. Compared with bare dendrimer and Lipofectamine 2000 (Lipo2000), NGO-PEG-dendrimer showed considerably lower cytotoxicity and higher transfection efficiency. As demonstrated by in vitro bioluminescence imaging and Western blotting assays, NGO-PEG-dendrimer effectively delivered anti-miR-21 into the cytoplasm and resulted in the upregulation of luciferase intensity and PTEN target protein expression in a dose-dependent manner. Moreover, transfection with anti-miR-21 by NGO-PEG-dendrimer led to stronger inhibition of cell migration and invasion than did bare dendrimer or Lipo2000 transfection. The intravenous delivery of anti-miR-21 via NGO-PEG-dendrimer induced a significant increase in the bioluminescence signal within the Fluc-3xPS reporter-transplanted tumor areas. These results suggest that NGO-PEG-dendrimer could be an efficient and a potential nanocarrier for delivering RNA oligonucleotides. In addition, the strategy of combining NGO-PEG-dendrimer with an activatable luciferase reporter allows the image-guided monitoring of the delivery process, which can provide insights into the RNA

  1. Repair of segmental bone defects with bone marrow and BMP-2 adenovirus in the rabbit radius

    NASA Astrophysics Data System (ADS)

    Cheng, Lijia; Lu, Xiaofeng; Shi, Yujun; Li, Li; Xue, Jing; Zhang, Li; Xia, Jie; Wang, Yujia; Zhang, Xingdong; Bu, Hong

    2012-12-01

    Bone tissue engineering (BTE) is approached via implantation of autogenous mesenchymal stem cells (MSCs), marrow cells, or platelet-rich plasma, etc. To the contrary, gene therapy combining with the bone marrow (BM) has not been often reported. This study was performed to investigate whether a modified BTE method, that is, the BM and a recombinant human bone morphogenetic protein-2 adenovirus (Ad.hBMP-2) gene administering in hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramics could accelerate the healing of segmental defects in the rabbit radius. In our study, ceramics were immersed in the adenovirus overnight, and half an hour before surgery, autologous BM aspirates were thoroughly mixed with the ceramics; at the same time, a 15-mm radius defect was introduced in the bilateral forelimbs of all animals, after that, this defect was filled with the following: (1) Ad.hBMP-2 + HA/β-TCP + autologous BM (group 1); (2) HA/β-TCP + Ad.hBMP-2 (group 2); (3) HA/β-TCP alone (group 3); (4) an empty defect as a control (group 4). Histological observation and μ-CT analyses were performed on the specimens at weeks 2, 4, 8, and 12, respectively. In group 1, new bone was observed at week 4 and BM appeared at week 12, in groups 2 and 3, new bone was observed at week 8 and it was more mature at week 12, in contrast, the defect was not bridged in group 4 at week 12. The new bone area percentage in group 1 was significantly higher than that in groups 2 and 3. Our study indicated that BM combined with hBMP-2 adenovirus and porous ceramics could significantly increase the amount of newly formed bone. And this modified BTE method thus might have potentials in future clinical application.

  2. 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.

  3. 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

  4. Targeting Motor End Plates for Delivery of Adenoviruses: An Approach to Maximize Uptake and Transduction of Spinal Cord Motor Neurons.

    PubMed

    Tosolini, Andrew Paul; Morris, Renée

    2016-01-01

    Gene therapy can take advantage of the skeletal muscles/motor neurons anatomical relationship to restrict gene expression to the spinal cord ventral horn. Furthermore, recombinant adenoviruses are attractive viral-vectors as they permit spatial and temporal modulation of transgene expression. In the literature, however, several inconsistencies exist with regard to the intramuscular delivery parameters of adenoviruses. The present study is an evaluation of the optimal injection sites on skeletal muscle, time course of expression and mice's age for maximum transgene expression in motor neurons. Targeting motor end plates yielded a 2.5-fold increase in the number of transduced motor neurons compared to injections performed away from this region. Peak adenoviral transgene expression in motor neurons was detected after seven days. Further, greater numbers of transduced motor neurons were found in juvenile (3-7 week old) mice as compared with adults (8+ weeks old). Adenoviral injections produced robust transgene expression in motor neurons and skeletal myofibres. In addition, dendrites of transduced motor neurons were shown to extend well into the white matter where the descending motor pathways are located. These results also provide evidence that intramuscular delivery of adenovirus can be a suitable gene therapy approach to treat spinal cord injury. PMID:27619631

  5. 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.

  6. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  7. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

    PubMed Central

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

    2015-01-01

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

  8. Surgical molecular navigation with a Ratiometric Activatable Cell Penetrating Peptide improves intraoperative identification and resection of small salivary gland cancers

    PubMed Central

    Hussain, Timon; Savariar, Elamprakash N.; Diaz-Perez, Julio A.; Messer, Karen; Pu, Minya; Tsien, Roger Y.; Nguyen, Quyen T.

    2015-01-01

    Background We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model. Methods Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time and tumor free survival were compared. Results RACPP guidance aided tumor detection (positive histology in 90% (27/30) vs. 48% (15/31) for WL, p<0.001). A ~25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded >90% detection sensitivity and specificity. Operating time was reduced by 54% (p<0.001), tumor free survival was increased with RACPP guidance (p=0.025). Conclusions RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications. PMID:25521629

  9. The construction and in vitro testing of photo-activatable cancer targeting folated anti-CD3 conjugates

    SciTech Connect

    Thompson, Stephen Dessi, John; Self, Colin H.

    2008-02-08

    The construction and in vitro testing of a photo-activatable anti-tumour immuno-regulatory antibody is described. In this 'cloaked' folated anti-CD3 antibody conjugate, the folate portion of the conjugate is free to bind to folate receptor expressing cancer cells, whilst the anti-CD3 activity is effectively rendered inert by a coating of photo-labile 2-nitrobenzyl groups. On irradiation with UV-A light the activity of the anti-CD3 antibody is restored, not only when it is required, but more importantly, only where it is required. The conjugate can then attract killer T-cells to the surface of the tumour cells and kill them. Unirradiated normal tissues, to which the conjugate has been targeted by specific and non-specific binding, remain unharmed. We believe that these 'photo-switchable' conjugates could be used to markedly improve the targeting of the immune response to folate receptor (FR) expressing ovarian and breast cancers whilst minimising the side effects in the rest of the body.

  10. Design of a simultaneous target and location-activatable fluorescent probe for visualizing hydrogen sulfide in lysosomes.

    PubMed

    Yang, Sheng; Qi, Yue; Liu, Changhui; Wang, Yijun; Zhao, Yirong; Wang, Lili; Li, Jishan; Tan, Weihong; Yang, Ronghua

    2014-08-01

    Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. The new synthesized fluorescent sensor, "SulpHensor", which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S, displays highly sensitive and selective fluorescence response to H2S under lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications. PMID:24975419

  11. Activatable fluorescent cys-diabody conjugated with indocyanine green derivative: consideration of fluorescent catabolite kinetics on molecular imaging

    PubMed Central

    Sano, Kohei; Nakajima, Takahito; Ali, Towhid; Bartlett, Derek W.; Wu, Anna M.; Kim, Insook; Paik, Chang H.; Choyke, Peter L.

    2013-01-01

    Abstract. Antibody fragments including diabodies have more desirable pharmacokinetic characteristics than whole antibodies. An activatable optical imaging probe based on a cys-diabody targeting prostate-specific membrane antigen conjugated with the near-infrared fluorophore, indocyanine green (ICG), was designed such that it can only be activated when bound to the tumor, leading to high signal-to-background ratios. We employed short polyethylene glycol (PEG) linkers between the ICG and the reactive functional group (Sulfo-OSu group), resulting in covalent conjugation of ICG to the cys-diabody, which led to lower dissociation of ICG from cys-diabody early after injection, reducing hepatic uptake. However, unexpectedly, high and long-term fluorescence was observed in the kidneys, liver, and blood pool more than 1 h after injection of the cys-diabody PEG-ICG conjugate. A biodistribution study using I125-labeled cys-diabody-ICG showed immediate uptake in the kidneys followed by a rapid decrease, while gastric activity increased due to released radioiodine during rapid cys-diabody-ICG catabolism in the kidneys. To avoid this catabolic pathway, it would be preferable to use antibody fragments large enough not to be filtered through glomerulus or to conjugate the fragments with fluorescent dyes that are readily excreted into urine when cleaved from the cys-diabody to achieve high tumor-specific detection. PMID:23752742

  12. Role of P38 MAPK on MMP Activity in Photothrombotic Stroke Mice as Measured using an Ultrafast MMP Activatable Probe

    PubMed Central

    Chang, Di; Wang, Yuan-Cheng; Bai, Ying-Ying; Lu, Chun-Qiang; Xu, Ting-Ting; Zhu, Lei; Ju, Shenghong

    2015-01-01

    Matrix metalloproteinases (MMPs) exert a dual effect in ischemic stroke and thus represent an ideal target for detection and therapy. However, to date, all clinical trials of MMP inhibitors have failed, and alternative drug candidates and therapeutic targets are urgently required. Nonetheless, further investigations are limited by the lack of non-invasive imaging techniques. Here, we report a novel, fast and ultrasensitive MMP activatable optical imaging probe for the dynamic visualization of MMP activity in photothrombotic stroke mice. This probe provides a significant signal enhancement in as little as 15 min, with the highest signal intensity occurring at 1 h post-injection, and shows high sensitivity in measuring MMP activity alterations, which makes it specifically suitable for the real-time visualization of MMP activity and drug discovery in preclinical research. Moreover, using this probe, we successfully demonstrate that the regulation of the p38 mitogen-activated protein kinase (MAPK) signal pathway is capable of modulating MMP activity after stroke, revealing a novel regulatory mechanism of postischemic brain damage and overcoming the limitations of traditional therapeutic strategies associated with MMP inhibitors by using a non-invasive molecular imaging method. PMID:26581247

  13. Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters.

    PubMed

    Sallenave, J M; Xing, Z; Simpson, A J; Graham, F L; Gauldie, J

    1998-03-01

    This report describes the design and construction of three recombinant adenoviruses of serotype 5 (Ad5) expressing elafin (EL), also called elastase-specific inhibitor. Three promoters were chosen to drive the synthesis of elafin: the small (380 bp) human cytomegalovirus promoter (HCMV), the Ad2 major late promoter (MLP) and the mouse cytomegalovirus (MCMV) promoter. Human alveolar epithelial cells (A549), as well as rat and human primary pulmonary fibroblasts were infected with Ad5-HCMV-EL, Ad5-MLP-EL, Ad5-MCMV-EL and with the control Ad5-dl70/3. The MCMV promoter was the most efficient promoter in all cells studied. MLP was the least efficient promoter Intermediate between MCMV and MLP was HCMV which was able to induce significant amounts of elafin, particularly in human A549 cells. When compared in vivo in rat lungs, results were similar; MCMV was the only promoter which induced significant amounts of elafin as assessed by Northern blot analysis and ELISA, even with a low dose of virus (3 x 10(8) p.f.u.). Our data indicate that the MCMV promoter is the promoter of choice for the strong induction of adenovirus-mediated transgenes in the lung and suggest its suitability both in rodent experimental models and in humans for investigative and therapeutic purposes. PMID:9614555

  14. Adenovirus with p16 gene exerts antitumor effect on laryngeal carcinoma Hep2 cells.

    PubMed

    Yang, Zhengang; Hu, Jingxia; Li, Dajun; Pan, Xinliang

    2016-08-01

    Laryngeal cancer is an uncommon form of cancer. The tumor suppressor P16, known to be mutated or deleted in various types of human tumor, including laryngeal carcinoma, is involved in the formation and development of laryngeal carcinoma. It has been previously reported that the inactivation or loss of P16 is associated with the acquisition of malignant characteristics. The current study hypothesized that restoring wild‑type P16 activity into P16‑null malignant Hep2 cells may exert an antitumor effect. A recombinant adenovirus carrying the P16 gene (Ad‑P16) was used to infect and express high levels of P16 protein in P16‑null Hep2 cells. Cell proliferation and invasion assays and polymerase chain reaction were performed to evaluate the effects of the P16 gene on cell proliferation and the antitumor effect on Hep2 cells. The results demonstrated that the Hep2 cells infected with Ad‑P16 exhibited significantly reduced cell proliferation, invasion and tumor volume compared with untreated or control adenovirus cells. Furthermore, the expression of laryngeal carcinoma‑associated genes, EGFR, survivin and cyclin D1, were measured in Ad‑P16‑infected cells and were significantly reduced compared with control groups. The results of the current study demonstrate that restoring wild‑type P16 activity into P16-null Hep2 cells exerts an antitumor effect. PMID:27277704

  15. 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

  16. 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

  17. Tamoxifen improves cytopathic effect of oncolytic adenovirus in primary glioblastoma cells mediated through autophagy

    PubMed Central

    Ulasov, Ilya V.; Shah, Nameeta; Kaverina, Natalya V.; Lee, Hwahyang; Lin, Biaoyang; Lieber, Andre; Kadagidze, Zaira G.; Yoon, Jae-Guen; Schroeder, Brett; Hothi, Parvinder; Ghosh, Dhimankrishna; Baryshnikov, Anatoly Y.; Cobbs, Charles S.

    2015-01-01

    Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy. PMID:25738357

  18. Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways.

    PubMed

    Kinoshita, Nanako; Mizuno, Takeo; Yoshihara, Yoshihiro

    2002-03-01

    Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology. PMID:11923184

  19. New human adenovirus isolated from a renal transplant recipient: description and characterization of candiate adenovirus type 34.

    PubMed Central

    Hierholzer, J C; Atuk, N O; Gwaltney, J M

    1975-01-01

    An antigenically distinct adenovirus is described which was isolated in March 1972 from the urine of a 17-year-old Caucasian male who was experiencing fever after receiving a kidney transplant from a cadaver in February. The adenovirus could not be isolated in April from a pharyngeal swab which yielded cytomegalovirus. Complement-fixation, hemagglutination-inhibition, and/or serum-neutralization tests on sequential serum specimens from the patient confirmed that the adenovirus infection occurred during March and showed that infections with cytomegalovirus and respiratory syncytial virus also occurred during late March and April. The patient's persistent fever, for which other causes could not be found, may have been associated with one or more of these infections. Upper respiratory symptoms and lung involvement were not found during this period. Mild liver dysfunction during this time could not be clearly related to adenovirus infection because of the presence of multiple other causes. The adenovirus may have been latent in the donor kidney and become active in the new host as a consequence of immunological impairment. The adenovirus, purified by terminal dilution and plaque procedures, has antigenic, morphological, biophysical, host susceptibility, and hemagglutinating properties characteristic of adenovirus group IA. Buoyant densities in CsCl are 1.340 g/ml for the virion, 1.304 g/ml for the group CF antigen (hexon), 1.295 g/ml for the major soluble complete hemagglutinin (dodecon), and 1.206 g/ml for the minor soluble complete hemagglutinin (tentatively, fiber dimer). The virus does not cross-react in reciprocal hemagglutination-inhibition and serum-neutralization tests with antisera to adenovirus types 1 to 33. We propose this virus as candidate adenovirus type 34 (Compton). Images PMID:170313

  20. The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry

    PubMed Central

    Loustalot, Fabien

    2015-01-01

    ABSTRACT The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FKCAV), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FKCAV entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family. PMID:26136571

  1. A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

    PubMed Central

    Appleby, CE; Kingston, PA; David, A; Gerdes, CA; Umaña, P; Castro, MG; Lowenstein, PR; Heagerty, AM

    2010-01-01

    Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of β-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases β-galactosidase expression 90-fold in SMC in vitro and ≈40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies. PMID:12907954

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

    PubMed Central

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

    2015-01-01

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

  3. Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

    PubMed Central

    Tran, P L; Weinbach, J; Opolon, P; Linares-Cruz, G; Reynes, J P; Grégoire, A; Kremer, E; Durand, H; Perricaudet, M

    1997-01-01

    A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis. PMID:9045862

  4. Five genome sequences of subspecies B1 human adenoviruses associated with acute respiratory disease.

    PubMed

    Dehghan, Shoaleh; Liu, Elizabeth B; Seto, Jason; Torres, Sarah F; Hudson, Nolan R; Kajon, Adriana E; Metzgar, David; Dyer, David W; Chodosh, James; Jones, Morris S; Seto, Donald

    2012-01-01

    Five genomes of human subspecies B1 adenoviruses isolated from cases of acute respiratory disease have been sequenced and archived for reference. These include representatives of two prevalent genomic variants of HAdV-7, i.e., HAdV-7h and HAdV-7d2. The other three are HAdV-3/16, HAdV-16 strain E26, and HAdV-3+7 strain Takeuchi. All are recombinant genomes. Genomics and bioinformatics provide detailed views into the genetic makeup of these pathogens and insight into their molecular evolution. Retrospective characterization of particularly problematic older pathogens such as HAdV-7h (1987) and intriguing isolates such as HAdV-3+7 strain Takeuchi (1958) may provide clues to their phenotypes and serology and may suggest protocols for prevention and treatment. PMID:22158846

  5. Transient acute adrenal insufficiency associated with adenovirus serotype 40 infection

    PubMed Central

    Rai, Birendra; Ali, Muhammad; Kumar, Varun; Krebit, Ibraheem

    2014-01-01

    We present an instance of a 6-year-old boy who was admitted with adenovirus infection and developed transient acute adrenal insufficiency, which required supplementation with glucocorticoids and mineralocorticoids for 8 weeks. Adenovirus has got adrenotropic potential and can cause adrenal insufficiency. We could not find any similar reported case in medical literature. We hope our case would add to the existing knowledge of adenoviral complications in paediatric patients. PMID:24928932

  6. Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus.

    PubMed

    Matoq, Amr; Salahuddin, Asma

    2016-01-01

    Adenoviruses are a common cause of respiratory infection, pharyngitis, and conjunctivitis in infants and young children. They are known to cause hepatitis and liver failure in immunocompromised patients; they are a rare cause of hepatitis in immunocompetent patients and have been known to cause fulminant hepatic failure. We present a 23-month-old immunocompetent infant who presented with acute noncholestatic hepatitis, hypoalbuminemia, generalized anasarca, and pancytopenia secondary to adenovirus infection. PMID:27340581

  7. Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus

    PubMed Central

    Salahuddin, Asma

    2016-01-01

    Adenoviruses are a common cause of respiratory infection, pharyngitis, and conjunctivitis in infants and young children. They are known to cause hepatitis and liver failure in immunocompromised patients; they are a rare cause of hepatitis in immunocompetent patients and have been known to cause fulminant hepatic failure. We present a 23-month-old immunocompetent infant who presented with acute noncholestatic hepatitis, hypoalbuminemia, generalized anasarca, and pancytopenia secondary to adenovirus infection. PMID:27340581

  8. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

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

    2016-01-01

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

  9. Polymeric oncolytic adenovirus for cancer gene therapy

    PubMed Central

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

    2015-01-01

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

  10. Adenovirus infection of the large bowel in HIV positive patients.

    PubMed Central

    Maddox, A.; Francis, N.; Moss, J.; Blanshard, C.; Gazzard, B.

    1992-01-01

    AIMS: To describe the microscopic appearance of adenovirus infection in the large bowel of human immunodeficiency virus (HIV) positive patients with diarrhoea. METHODS: Large bowel biopsy specimens from 10 HIV positive patients, eight of whom were also infected with other gastrointestinal pathogens, with diarrhoea were examined, together with six small bowel biopsy specimens from the same group of patients. Eight of the patients had AIDS. The biopsy specimens were examined by light microscopy performed on haematoxylin and eosin stained and immunoperoxidase preparations, the latter using a commercially available antibody (Serotec MCA 489). Confirmation was obtained with electron microscopy. RESULTS: The morphological appearance of cells infected with adenovirus showed characteristic nuclear and cellular changes, although the inflammatory reaction was non-specific. Immunoperoxidase staining for adenovirus was sensitive and specific, and the presence of viral inclusions consistent with adenovirus was confirmed by electron microscopy. CONCLUSIONS: The light microscopic features of adenovirus infection are distinctive and immunocytochemistry with a commercially available antibody is a sensitive and specific means of confirming the diagnosis. Further studies of the role of adenovirus in causing diarrhoea in these patients are indicated. Images PMID:1401177

  11. Design and study of activatable ("OFF/ON") quantum dots (Qdots): ligand selection for Qdot surface modification for controlling Qdot fluorescence quenching and restoration

    NASA Astrophysics Data System (ADS)

    Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Mitra, Rajendra N.; Banerjee, Subhash; Santra, Swadeshmukul

    2012-03-01

    We report design and synthesis of a series of activatable "OFF/ON" CdS:Mn/ZnS quantum dot (Qdot) based sensing probes. The Qdot "OFF" state represent the "quenched state" where the Qdot fluorescence is quenched by ligands attached to Qdot surface. Fluorescence quenching is likely due to ligand assisted electron transfer process. Qdot fluorescence is restored when the electron transfer process is stopped. Using this activatable Qdots, we have successfully demonstrated usefulness of these Qdot probes for reliable detection of toxic cadmium ions in solution, selective detection of glutathione and sensitive detection of intracellular cancer drug release event. In this paper, we will discuss a simple but robust method of making water-soluble CdS:Mn/ZnS Qdots at the room-temperature. Two different water-soluble biomolecules, the N-acetyl cysteine (NAC) and the glutathione (GSH) were used as surface coating ligands. This is a singlestep, one-pot synthesis where the Qdot nanocrystals were grown in the presence of the biomolecules. These Qdots were characterized by fluorescence spectroscopy. Stability of the GSH coated Qdots and the NAC coated Qdots were studied by treating with ethylenediaminetetraacetic acid (EDTA, a strong chelating agent for Zn and Cd ions). Our results show that fluorescence properties of Qdots are affected by the type of surface coated ligands. In comparison to the GSH coated Qdots, the NAC coated Qdots show broad but strong emission towards near infra-red region. When treated with EDTA, fluorescence property of the GSH coated Qdot was affected less than the NAC coated Qdots. This preliminary study shows that NAC coated Qdots could potentially be used to develop activatable ("OFF/ON") probes for potential deep-tissue imaging applications. Similarly, the GSH coated Qdots could be applied for probing desired analytes or for bioimaging purposes in environmentally harsh conditions.

  12. Au@Ag/Au nanoparticles assembled with activatable aptamer probes as smart ``nano-doctors'' for image-guided cancer thermotherapy

    NASA Astrophysics Data System (ADS)

    Shi, Hui; Ye, Xiaosheng; He, Xiaoxiao; Wang, Kemin; Cui, Wensi; He, Dinggeng; Li, Duo; Jia, Xuekun

    2014-07-01

    Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo ``nano-doctors'' that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as the model, in vitro and in vivo studies of A549 lung cancer verified that the ATNP greatly improved imaging contrast and specific destruction, suggesting a robust and versatile theranostic strategy for personalized medicine in future.Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo ``nano-doctors'' that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as

  13. Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization.

    PubMed

    Gao, Wentao; Soloff, Adam C; Lu, Xiuhua; Montecalvo, Angela; Nguyen, Doan C; Matsuoka, Yumi; Robbins, Paul D; Swayne, David E; Donis, Ruben O; Katz, Jacqueline M; Barratt-Boyes, Simon M; Gambotto, Andrea

    2006-02-01

    The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza. PMID:16439551

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

    PubMed

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

    2016-02-01

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

  15. Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

    PubMed

    Zhang, Nian-Hua; Peng, Rui-Qing; Ding, Ya; Zhang, Xiao-Shi

    2016-08-01

    The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity. PMID:27373420

  16. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy. PMID:25674795

  17. Redoxable heteronanocrystals functioning magnetic relaxation switch for activatable T1 and T2 dual-mode magnetic resonance imaging.

    PubMed

    Kim, Myeong-Hoon; Son, Hye-Young; Kim, Ga-Yun; Park, Kwangyeol; Huh, Yong-Min; Haam, Seungjoo

    2016-09-01

    T1/T2 dual-mode magnetic resonance (MR) contrast agents (DMCAs) have gained much attention because of their ability to improve accuracy by providing two pieces of complementary information with one instrument. However, most of these agents are "always ON" systems that emit MR contrast regardless of their interaction with target cells or biomarkers, which may result in poor target-to-background ratios. Herein, we introduce a rationally designed magnetic relaxation switch (MGRS) for an activatable T1/T2 dual MR imaging system. Redox-responsive heteronanocrystals, consisting of a superparamagnetic Fe3O4 core and a paramagnetic Mn3O4 shell, are synthesized through seed-mediated growth and subsequently surface-modified with polysorbate 80. The Mn3O4 shell acts as both a protector of Fe3O4 in aqueous environments to attenuate T2 relaxation and as a redoxable switch that can be activated in intracellular reducing environments by glutathione. This simultaneously generates large amounts of magnetically decoupled Mn(2+) ions and allows Fe3O4 to interact with the water protons. This smart nanoplatform shows an appropriate hydrodynamic size for the EPR effect (10-100 nm) and demonstrates biocompatibility. Efficient transitions of OFF/ON dual contrast effects are observed by in vitro imaging and MR relaxivity measurements. The ability to use these materials as DMCAs is demonstrated via effective passive tumor targeting for T1- and T2-weighted MR imaging in tumor-bearing mice. PMID:27281684

  18. Activatable hyaluronic acid nanoparticle as a theranostic agent for optical/photoacoustic image-guided photothermal therapy.

    PubMed

    Zhang, Liwen; Gao, Shi; Zhang, Fan; Yang, Kai; Ma, Qingjie; Zhu, Lei

    2014-12-23

    Photothermal therapy (PTT) is an emerging treatment modality that is under intensive preclinical investigations for the treatment of various medical conditions, including cancer. However, the lack of targeting function of PTT agents hampers its clinical application. An effective and nontoxic delivery vehicle that can carry PTT agents into tumor areas is still needed urgently. In this study, we developed a multifunctional nanocomposite by loading copper sulfide (CuS) into Cy5.5-conjugated hyaluronic acid nanoparticles (HANP), obtaining an activatable Cy5.5-HANP/CuS (HANPC) nanocomposite. In this system, Cy5.5 fluorescent signal is quenched by CuS inside the particle until the whole nanocomposite is degraded by hyaluronidase present in tumor, giving strong fluorescence signals delineating the tumor. Importantly, CuS with strong NIR absorbance appears to be an excellent contrast agent for photoacoustic (PA) imaging and an effective PTT agent. After intravenous administration of HANPC into SCC7 tumor-bearing mice, high fluorescence and PA signals were observed in the tumor area over time, which peaked at the 6 h time point (tumor-to-normal tissue ratio of 3.25±0.25 for optical imaging and 3.8±0.42 for PA imaging). The tumors were then irradiated with a laser, and a good tumor inhibition rate (89.74% on day 5) was observed. Our studies further encourage application of this HA-based multifunctional nanocomposite for image-guided PTT in biomedical applications, especially in cancer theranostics. PMID:25402600

  19. Novel 19F Activatable Probe for the Detection of Matrix Metalloprotease-2 Activity by MRI/MRS

    PubMed Central

    2015-01-01

    Matrix metalloproteases (MMPs) have been found to be highly expressed in a variety of malignant tumor tissues. Noninvasive visualization of MMP activity may play an important role in the diagnosis of MMP associated diseases. Here we report the design and synthesis of a set of fluorine-19 dendron-based magnetic resonance imaging (MRI) probes for real-time imaging of MMP-2 activity. The probes have the following features: (a) symmetrical fluorine atoms; (b) the number of fluorine atoms can be increased through facile chemical modification; (c) readily accessible peptide sequence as the MMP-2 substrate; (d) activatable 19F signal (off/on mode) via paramagnetic metal ion incorporation. Following optimization for water solubility, one of the probes was selected to evaluate MMP-2 activity by 19F magnetic resonance spectroscopy (MRS). Our results showed that the fluorine signal increased by 8.5-fold in the presence of MMP-2. The specific cleavage site was verified by mass spectrometry. The selected probe was further applied to detect secreted MMP-2 activity of living SCC7 squamous cell carcinoma cells. The fluorine signal was increased by 4.8-fold by MRS analysis after 24 h incubation with SCC7 cells. This type of fluorine probe can be applied to evaluate other enzyme activities by simply tuning the substrate structures. This symmetrical fluorine dendron-based probe design extends the scope of the existing 19F MRI agents and provides a simple but robust method for real-time 19F MRI application. PMID:25271556

  20. Preclinical evaluation of light-activatable, bispecific anti-human CD3 antibody conjugates as anti-ovarian cancer therapeutics

    PubMed Central

    Dessi, John

    2009-01-01

    The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the ‘cloaked’ conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients. PMID:20068406

  1. Thrombin-activatable fibrinolysis inhibitor activity and global fibrinolytic capacity in Type 1 diabetes: evidence for normal fibrinolytic state.

    PubMed

    Harmanci, Ayla; Kandemir, Nurgun; Dagdelen, Selcuk; Gonc, Nazli; Buyukasik, Yahya; Alikasifoglu, Ayfer; Kirazli, Serafettin; Ozon, Alev; Gurlek, Alper

    2006-01-01

    Hypofibrinolysis is a state that is commonly observed in type 2 diabetic patients, a finding also possibly related to obesity and insulin resistance. There is little information, however, regarding the status of fibrinolytic system in Type 1 diabetes, in particular as reflected by thrombin-activatable fibrinolysis inhibitor (TAFI) activity and global fibrinolytic capacity (GFC). To provide information in this respect, 30 Type 1 diabetic patients (median age=16) and 28 healthy controls (median age=14) were enrolled in this study. The median duration of diabetes was 7 years, and median HbA(1c) was 8.85% (range: 5.5-11.9%) in the diabetic group. None of the patients had macrovascular complications. Microvascular complications were present in a total of eight patients (nephropathy: n=5; retinopathy: n=3). A comparison of the TAFI activity between the patient (median 84.9, range: 71.5-103.3%) and the control groups (median=83.3, range: 63.7-97.4%) yielded no statistically significant difference (P=.950). Similarly, GFC was comparable between the two groups (median=8.22, range: 0.72-22.38 microg/ml, and median=13.32, range: 3.0-23.22 microg/ml, respectively, in the diabetic and control groups, P=.086). TAFI activity did not significantly correlate with age, albumin excretion, fasting plasma glucose, HbA(1c), D-dimer, and fibrinogen by Spearman rank correlation test. There was as a significant inverse correlation between GFC and TAFI activity (r=-.414, P=.006). Contrary to the previous observations in Type 2 diabetes, our data suggest that fibrinolytic activity is not adversely affected by Type 1 diabetes, and it has no relationship with the degree of metabolic control. PMID:16389166

  2. Association between thrombin-activatable fibrinolysis inhibitor gene polymorphisms and venous thrombosis risk: a meta-analysis.

    PubMed

    Wang, Wei; Ma, He; Lu, Lili; Sun, Guixiang; Liu, Dang; Zhou, Yunti; Tong, Yue; Lu, Zhaojun

    2016-06-01

    Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important antifibrinolytic factor that has been shown in increased concentrations to be associated with an increased risk for venous thrombosis. However, the effect of TAFI gene polymorphisms on the risk of venous thrombosis remains debatable. The aim of the current study was to evaluate the association of three single nucleotide polymorphisms: 505G>A (rs3742264), 1040 C>T (rs1926447) and -438G>A (rs2146881) with venous thrombosis risk using a meta-analysis. A systematic literature search for eligible studies published before 20 January 2015 was conducted in PubMed, EMBASE, Web of Science, WanFang database and Chinese National Knowledge Infrastructure. We assessed the possible association by pooled odds ratio and its 95% confidence interval. A total of 14 independent case-control studies including 2970 cases and 3049 controls were enrolled in the final meta-analysis. A significant reduction of venous thrombosis risk in the 505G>A polymorphism was observed under allele comparison, homozygote comparison and recessive models, but opposite results were seen in Asians. Likewise, there was a significant decreased susceptibility to venous thrombosis in the 1040C>T polymorphism in homozygote comparison and recessive models. In the subgroup analysis, the nonvenous thromboembolism disease group showed a significantly increased venous thrombosis risk. Pooled estimates did not show evidence of association between -438G>A and venous thrombosis risk in any genetic model. This meta-analysis suggested that although the -438G>T polymorphism is not correlated with venous thrombosis risk in all models, a trend toward reduced risk still could be observed. The A allele and AA genotype of 505G>A in whites and the TT genotype of 1040C>T were significantly associated with a decreased risk of venous thrombosis, except in the non-venous thromboembolism group. PMID:26656901

  3. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6. PMID:27126696

  4. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... dilution in a varying serum-constant virus neutralization test using 50 to 300 TCID50 of canine adenovirus... virus neutralization test using 50 to 300 TCID50 of canine adenovirus. (i) A geometric mean titer of...

  5. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... dilution in a varying serum-constant virus neutralization test using 50 to 300 TCID50 of canine adenovirus... virus neutralization test using 50 to 300 TCID50 of canine adenovirus. (i) A geometric mean titer of...

  6. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... dilution in a varying serum-constant virus neutralization test using 50 to 300 TCID50 of canine adenovirus... virus neutralization test using 50 to 300 TCID50 of canine adenovirus. (i) A geometric mean titer of...

  7. SCREENING STUDIES TO DETRMINE THE EFFECTIVENESS OF CHLORINE TO INACTIVATE ADENOVIRUS (RM.C.M.4)

    EPA Science Inventory

    To evaluate the susceptibility of adenovirus (CCL organism) to inactivation by chemical disinfectants, including chlorine and chloramine. Bench scale disinfection studies will be conducted on adenovirus and selected bacteriophages suspended in oxidant demand free buffered water: ...

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

    PubMed Central

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

    2015-01-01

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

  9. Functional dissection of adenovirus VAI RNA.

    PubMed Central

    Furtado, M R; Subramanian, S; Bhat, R A; Fowlkes, D M; Safer, B; Thimmappaya, B

    1989-01-01

    During the course of adenovirus infection, the VAI RNA protects the translation apparatus of host cells by preventing the activation of host double-stranded RNA-activated protein kinase, which phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2. In the absence of VAI RNA, protein synthesis is drastically inhibited at late times in infected cells. The experimentally derived secondary structure of VAI RNA consists of two extended base-paired regions, stems I and III, which are joined by a short base-paired region, stem II, at the center. Stems I and II are joined by a small loop, A, and stem III contains a hairpin loop, B. At the center of the molecule and at the 3' side, stems II and III are connected by a short stem-loop (stem IV and hairpin loop C). A fourth, minor loop, D, exists between stems II and IV. To determine sequences and domains critical for function within this VAI RNA structure, we have constructed adenovirus mutants with linker-scan substitution mutations in defined regions of the molecule. Cells infected with these mutants were analyzed for polypeptide synthesis, virus yield, and eIF-2 alpha kinase activity. Our results showed that disruption of base-paired regions in the distal parts of the longest stems, I and III, did not affect function, whereas mutations causing structural perturbations in the central part of the molecule containing stem II, the proximal part of stem III, and the central short stem-loop led to loss of function. Surprisingly, one substitution mutant, sub742, although dramatically perturbing the integrity of the structure of this central portion, showed a wild-type phenotype, suggesting that an RNA with an alternate secondary structure is functional. On the basis of sensitivity to single-strand-specific RNases, we can derive a novel secondary structure for the mutant RNA in which a portion of the sequences may fold to form a structure that resembles the central part of the wild-type molecule

  10. 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

  11. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence–Quencher Probe as a Tool for Functional Antibody Screening

    PubMed Central

    Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D.; Hiraga, Kaori

    2015-01-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody–drug conjugates. Here we describe a novel activatable fluorescence–quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  12. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    PubMed

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

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

    PubMed Central

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

    2015-01-01

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

  14. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Canine Hepatitis and Canine Adenovirus... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  15. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  16. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  17. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

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

    EPA Science Inventory

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

  19. Physical organization of subgroup B human adenovirus genomes.

    PubMed Central

    Tibbetts, C

    1977-01-01

    Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved. Images PMID:916027

  20. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle. PMID:25498212

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

    PubMed

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

    2009-10-01

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

  2. Human adenovirus: Viral pathogen with increasing importance

    PubMed Central

    2014-01-01

    The aim of this review is to describe the biology of human adenovirus (HAdV), the clinical and epidemiological characteristics of adenoviral epidemic keratoconjunctivitis and to present a practical update on its diagnosis, treatment, and prophylaxis. There are two well-defined adenoviral keratoconjunctivitis clinical syndromes: epidemic keratoconjunctivitis (EKC) and pharyngoconjunctival fever (PCF), which are caused by different HAdV serotypes. The exact incidence of adenoviral conjunctivitis is still poorly known. However, cases are more frequent during warmer months. The virus is endemic in the general population, and frequently causes severe disease in immunocompromised patients, especially the pediatric patients. Contagion is possible through direct contact or fomites, and the virus is extremely resistant to different physical and chemical agents. The clinical signs or symptoms of conjunctival infection are similar to any other conjunctivitis, with a higher incidence of pseudomembranes. In the cornea, adenoviral infection may lead to keratitis nummularis. Diagnosis is mainly clinical, but its etiology can be confirmed using cell cultures, antigen detection, polymerase chain reaction or immunochromatography. Multiple treatments have been tried for this disease, but none of them seem to be completely effective. Prevention is the most reliable and recommended strategy to control this contagious infection. PMID:24678403

  3. Adenovirus 36 and Obesity: An Overview

    PubMed Central

    Ponterio, Eleonora; Gnessi, Lucio

    2015-01-01

    There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed. PMID:26184280

  4. Enhanced expression of adenovirus transforming proteins.

    PubMed Central

    Gaynor, R B; Tsukamoto, A; Montell, C; Berk, A J

    1982-01-01

    Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated. Images PMID:7143568

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

    PubMed

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

    2016-01-01

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

  6. Theranostic nanoparticles for enzyme-activatable fluorescence imaging and photodynamic/chemo dual therapy of triple-negative breast cancer

    PubMed Central

    Choi, Jaehee; Kim, Hyunjin

    2015-01-01

    Background Triple-negative breast cancer (TNBC) is a highly diverse group of cancers characterized by tumors that does not express estrogen and progesterone receptors, as well as human epidermal growth factor receptor 2 (HER2) gene expression. TNBC is associated with poor prognosis due to high rate of recurrence and distance metastasis, lack of response to hormonal or HER2-targeted therapies, and partial response to chemotherapy. Hence, development of new therapeutic strategies to overcome such limitations is of great importance. Here we describe the application of photosensitizer-conjugated and camptothecin (CPT)-encapsulated hyaluronic acid (HA) nanoparticles as enzyme-activatable theranostic nanoparticles (EATNP) for near-infrared (NIR) fluorescence imaging and photodynamic/chemo dual therapy of TNBC. Methods For the preparation of EATNPs, chlorin e6 (Ce6), a second generation photosensitizer, was covalently conjugated to a monomethoxy poly(ethylene glycol)-grafted HA backbone. Ce6-conjugated HA (Ce6-HA) formed self-assembled nanoparticles (i.e., Ce6-HA NPs) in an aqueous solution. Subsequently, CPT, a topoisomerase 1 inhibitor with remarkable anticancer efficacy but with low water solubility, was encapsulated inside the hydrophobic core of Ce6-HA NPs thereby forming EATNPs. Results Fluorescence and singlet oxygen generation (SOG) of EATNPs are quenched in its native state. Treatment of EATNPs with hyaluronidase (HAdase) induces enzyme concentration-dependent activation of NIR fluorescence and SOG. Moreover, HAdase-mediated degradation of the nanoparticles also triggers the release of CPT from the EATNPs. In vitro confocal microscopy and cytotoxicity tests confirmed that EATNPs were efficiently introduced into MDA-MB-231 TNBC cell line, thereby inducing better cytotoxicity than that by free CPT. Additional light irradiation onto the EATNP-treated cells significantly increased therapeutic efficacy in TNBC, which indicates that EATNP plays an important role in

  7. RECOMBINANT SWINE INTERFERON BETA PROTECTS SWINE ALVEOLAR MACROPHAGES AND MARC 145 CELLS FROM INFECTION WITH PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Swine interferon beta (swIFN beta) produced in 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN beta gene was tested for antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). Marc145 cells were incubated overni...

  8. Disseminated adenovirus infection in an immunocompromised host. Pitfalls in diagnosis.

    PubMed

    Landry, M L; Fong, C K; Neddermann, K; Solomon, L; Hsiung, G D

    1987-09-01

    In this report, a bone marrow transplant recipient with rapidly fatal gastroenteritis is presented. The presence of intranuclear inclusions on postmortem light microscopic examination of liver, lung, and small bowel tissue was considered diagnostic of cytomegalovirus infection. However, electron microscopic examination of liver tissue demonstrated adenovirus infection. This was confirmed by isolation of an adenovirus type 2 with unusual laboratory features from liver, lung, colon contents, serum, esophageal swab, and oral ulcerations. Results of a complement fixation test for antibodies to adenovirus performed on postmortem serum samples were negative, and a titer of 1:4 was noted for antibody against cytomegalovirus. This case illustrates the diagnostic pitfalls that may be encountered in establishing a specific viral diagnosis in severely ill patients. PMID:2821806

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

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

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

  10. Species-Specific Identification of Human Adenoviruses in Sewage.

    PubMed

    Wieczorek, Magdalena; Krzysztoszek, Arleta; Witek, Agnieszka

    2015-01-01

    Human adenovirus (HAdV) diversity in sewage was assessed by species-specific molecular methods. Samples of raw sewage were collected in 14 sewage disposal systems from January to December 2011, in Poland. HAdVs were detected in 92.1% of the analysed sewage samples and was significantly higher at cities of over 100 000 inhabitants. HAdV DNA was detected in sewage during all seasons. The most abundant species identified were HAdV-F (average 89.6%) and -A (average 19.6%), which are associated with intestine infections. Adenoviruses from B species were not detected. The result of the present study demonstrate that human adenoviruses are consistently present in sewage in Poland, demonstrating the importance of an adequate treatment before the disposal in the environment. Multiple HAdV species identified in raw sewage provide new information about HAdV circulation in the Polish population. PMID:26094312

  11. 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

  12. 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

  13. Crystal Structure of Enteric Adenovirus Serotype 41 Short Fiber Head

    PubMed Central

    Seiradake, Elena; Cusack, Stephen

    2005-01-01

    Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding. PMID:16254343

  14. The Prevalence of Rotavirus and Adenovirus in the Childhood Gastroenteritis

    PubMed Central

    Ozsari, Tamer; Bora, Gulhan; Kaya, Bulent; Yakut, Kahraman

    2016-01-01

    Background Acute gastroenteritis stemming from viral causes is very common during the childhood period. Rotavirus and enteric adenovirus are the most common factors of acute gastroenteritis encountered in infants and children. However, the epidemiology of rotavirus and enteric adenovirus gastroenteritis in the east Anatolia region is not well-known. Objectives We aimed to evaluate the relationship between the distribution of antigen positivity in rotavirus and enteric adenovirus antigen tests required cases and demographic data retrospectively in pediatric patients admitted to our hospital. Patients and Methods The records of stool sample analyses for 1154 patients admitted to our hospital from June 2011 to December 2011 with complaints of diarrhea were retrospectively examined. The presence of rotavirus and enteric adenovirus antigens in stool specimens was investigated by means of an immunochromatographic test. Results Viral antigens were detected in 327 (28.3%) stool specimens out of 1154. Among the positive results, the frequency was 73.7% for rotavirus and 26.2% for adenovirus. While the detected rotavirus antigen rate was high for all age groups, it was highest for children under the age of 2, with a rate of 57.1%. Moreover, the rotavirus infections were observed at a rate of 44.3% in winter and of 24.6% in autumn. Conclusions The most important factor in childhood acute gastroenteritis in east Anatolia is the rotavirus. Rotavirus and adenovirus antigens should be routinely investigated as a factor in fresh stool samples for the accurate diagnosis and treatment of gastroenteritis in children in the winter and autumn months.

  15. Vault nanoparticles containing an adenovirus-derived membrane lytic protein facilitate toxin and gene transfer.

    PubMed

    Lai, Cheng-Yu; Wiethoff, Chris M; Kickhoefer, Valerie A; Rome, Leonard H; Nemerow, Glen R

    2009-03-24

    Nonviral methods of gene delivery possess several advantages over that of viral-based vectors, including having increased safety. However, the ability to achieve effective transport of therapeutic molecules across host cell membranes via nonviral methods remains a significant goal. Cell-derived nanoparticles known as vaults have been proposed as novel candidate transfer vehicles for various foreign molecules. Recombinant vault particles enter cells via macropinocytosis or phagocytosis but lack demonstrable membrane penetrating activity. To explore the feasibility of improving vault penetration into target cells, we incorporated the membrane lytic domain of adenovirus protein VI (pVI) into the interior of recombinant vault particles via fusion to the vault poly(ADP-ribose) polymerase (VPARP) interaction domain. The membrane lytic activity of the pVI domain was retained upon incorporation into vault particles. Moreover, internalization of vault-pVI complexes into murine macrophages promoted co-delivery of a soluble ribotoxin or a cDNA plasmid encoding GFP. These findings indicate that vault particles can be modified to enhance cell transfer of selected biomolecules. PMID:19226129

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

    PubMed

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

    2008-10-01

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

  17. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

    PubMed Central

    Caruso, M; Pham-Nguyen, K; Kwong, Y L; Xu, B; Kosai, K I; Finegold, M; Woo, S L; Chen, S H

    1996-01-01

    Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma. Images Fig. 5 PMID:8876130

  18. Functional prediction of hypothetical proteins in human adenoviruses.

    PubMed

    Dorden, Shane; Mahadevan, Padmanabhan

    2015-01-01

    Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins. PMID:26664031

  19. 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

  20. Human Adenovirus Type 2 but Not Adenovirus Type 12 Is Mutagenic at the Hypoxanthine Phosphoribosyltransferase Locus of Cloned Rat Liver Epithelial Cells

    PubMed Central

    Paraskeva, Christos; Roberts, Carl; Biggs, Paul; Gallimore, Phillip H.

    1983-01-01

    Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose. PMID:6572280

  1. Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

    SciTech Connect

    Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E.; Leeuwen, M. van; Boheemen, S. van; Jong, A.A.W. de; Osterhaus, A.D.M.E.; Smits, S.L.; Kuiken, T.

    2013-05-25

    Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. - Highlights: ► Lesions typical for adenovirus infection detected in cloacal bursa of dead gulls. ► Confirmation of adenovirus infection by electron microscopy and deep sequencing. ► Sequence analysis indicates that it is a novel adenovirus in the genus Aviadenovirus. ► The novel (Gull) adenovirus was detected in multiple organs of two species of gulls.

  2. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    PubMed

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T(1) protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2005-01-01

    Eighty-eight adenovirus (Ad) isolates and associated clinical data were collected from walk-in patients with influenza-like illness in Egypt during routine influenza surveillance from 1999 through 2002. Respiratory Ad distributions are geographically variable, and serotype prevalence has not been previously characterized in this region. Serotype identity is clinically relevant because it predicts vaccine efficacy and correlates strongly with both clinical presentation and epidemiological pattern. Species and serotype identities were determined using several well-validated multiplex PCR protocols culled from the literature and supplemented with a few novel primer sets designed to identify rare types. The isolates included common species B1 serotypes (Ad3 and Ad7), common species C serotypes (Ad1, Ad2, and Ad5), the less common species B2 serotype Ad11, and three isolates of the rare species B1 serotype Ad16. Two isolates that appear to be variant Ad16 were also identified. Fifteen coinfections of multiple adenoviral types, primarily AdB/AdC and Ad3/Ad7 dual infections, were detected. The majority of these were verified using redundant PCR tests targeted at multiple genes. PCR is able to resolve coinfections, in contrast to traditional serum neutralization tests. PCR is also comparatively rapid and requires very little equipment. Application of the method allowed an inclusive determination of the serotypes found in the Egyptian respiratory sample set and demonstrated that coinfections are common and may play a previously unrecognized role in adenovirus pathogenesis, evolution, and epidemiology. In particular, coinfections may influence adenoviral evolution, as interserotypic recombination has been identified as a source of emerging strains. PMID:16272512

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Counihan, Katrina L; Skerratt, Lee F; Franson, J Christian; Hollmén, Tuula E

    2015-11-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections. PMID:26342465

  7. Transport of human adenoviruses in porous media

    NASA Astrophysics Data System (ADS)

    Kokkinos, Petros; Syngouna, Vasiliki I.; Tselepi, Maria A.; Bellou, Maria; Chrysikopoulos, Constantinos V.; Vantarakis, Apostolos

    2015-04-01

    Groundwater may be contaminated with infective human enteric viruses from various wastewater discharges, sanitary landfills, septic tanks, agricultural practices, and artificial groundwater recharge. Coliphages have been widely used as surrogates of enteric viruses, because they share many fundamental properties and features. Although a large number of studies focusing on various factors (i.e. pore water solution chemistry, fluid velocity, moisture content, temperature, and grain size) that affect biocolloid (bacteria, viruses) transport have been published over the past two decades, little attention has been given toward human adenoviruses (hAdVs). The main objective of this study was to evaluate the effect of pore water velocity on hAdV transport in water saturated laboratory-scale columns packed with glass beads. The effects of pore water velocity on virus transport and retention in porous media was examined at three pore water velocities (0.39, 0.75, and 1.22 cm/min). The results indicated that all estimated average mass recovery values for hAdV were lower than those of coliphages, which were previously reported in the literature by others for experiments conducted under similar experimental conditions. However, no obvious relationship between hAdV mass recovery and water velocity could be established from the experimental results. The collision efficiencies were quantified using the classical colloid filtration theory. Average collision efficiency, α, values decreased with decreasing flow rate, Q, and pore water velocity, U, but no significant effect of U on α was observed. Furthermore, the surface properties of viruses and glass beads were used to construct classical DLVO potential energy profiles. The results revealed that the experimental conditions of this study were unfavorable to deposition and that no aggregation between virus particles is expected to occur. A thorough understanding of the key processes governing virus transport is pivotal for public

  8. Adenovirus Dodecahedron, as a Drug Delivery Vector

    PubMed Central

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

    2009-01-01

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

  9. Immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying B and T cell epitopes of foot-and-mouth disease.

    PubMed

    Pan, Qunxing; Wang, Hui; Ouyang, Wei; Wang, Xiaoli; Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; He, Kongwang

    2016-01-20

    Virus-like particles (VLPs) vaccines combine many of the advantages of whole-virus vaccines and recombinant subunit vaccines, integrating key features that underlay their immunogenicity, safety and protective potential. We have hypothesized here the effective insertion of the VP1 epitopes (three amino acid residues 21-40, 141-160 and 200-213 in VP1, designated VPe) of foot-and-mouth disease (FMDV) within the external loops of PPV VP2 could be carried out without altering assembly based on structural and antigenic data. To investigate the possibility, development of two recombinant adenovirus rAd-PPV:VP2-FMDV:VPe a or rAd-PPV:VP2-FMDV:VPe b were expressed in HEK-293 cells. Out of the two insertion strategies tested, one of them tolerated an insert of 57 amino acids in one of the four external loops without disrupting the VLPs assembly. Mice were inoculated with the two recombinant adenoviruses, and an immunogenicity study showed that the highest levels of FMDV-specific humoral responses and T cell proliferation could be induced by rAd-PPV:VP2-FMDV:VPe b expressing hybrid PPV:VLPs (FMDV) in the absence of an adjuvant. Then, the protective efficacy of inoculating swine with rAd-PPV:VP2-FMDV:VPe b was tested. All pigs inoculated with rAd-PPV:VP2-FMDV:VPe b were protected from viral challenge, meanwhile the neutralizing antibody titers were significantly higher than those in the group inoculated with swine FMD type O synthetic peptide vaccine. Our results clearly demonstrate the potential usefulness of adenovirus-derived PPV VLPs as a vaccine strategy in prevention of FMDV. PMID:26685093

  10. Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

    PubMed

    Choi, Jin Huk; Schafer, Stephen C; Freiberg, Alexander N; Croyle, Maria A

    2015-08-01

    The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus. PMID:25549696

  11. Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

    SciTech Connect

    Rhee, Juong G.; Li, Daqing; Suntharalingam, Mohan; Guo Chuanfa; O'Malley, Bert W.; Carney, James P. . E-mail: jcarney@som.umaryland.edu

    2007-01-01

    Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.

  12. Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.

    PubMed

    Itoh, M; Masuda, K; Ito, Y; Akizawa, T; Yoshioka, M; Imai, K; Okada, Y; Sato, H; Seiki, M

    1996-04-01

    Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors. PMID:8743567

  13. Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

    PubMed Central

    Behr, Michael; Kaufmann, Johanna K.; Ketzer, Patrick; Engelhardt, Sarah; Mück-Häusl, Martin; Okun, Pamela M.; Petersen, Gabriele; Neipel, Frank; Hassel, Jessica C.; Ehrhardt, Anja; Enk, Alexander H.; Nettelbeck, Dirk M.

    2014-01-01

    Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis. PMID:24760010

  14. 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

  15. Bioaccumulation of animal adenoviruses in the pink shrimp.

    PubMed

    Luz, Roger B; Staggemeier, Rodrigo; Fabres, Rafael B; Soliman, Mayra C; Souza, Fernanda G; Gonçalves, Raoni; Fausto, Ivone V; Rigotto, Caroline; Heinzelmann, Larissa S; Henzel, Andréia; Fleck, Juliane D; Spilki, Fernando R

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems. PMID:26413052

  16. Bioaccumulation of animal adenoviruses in the pink shrimp

    PubMed Central

    Luz, Roger B.; Staggemeier, Rodrigo; Fabres, Rafael B.; Soliman, Mayra C.; Souza, Fernanda G.; Gonçalves, Raoni; Fausto, Ivone V.; Rigotto, Caroline; Heinzelmann, Larissa S.; Henzel, Andréia; Fleck, Juliane D.; Spilki, Fernando R.

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems. PMID:26413052

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

    PubMed

    Weaver, Eric A; Barry, Michael A

    2013-01-01

    Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5) are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10(8) virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications. PMID:23991187

  18. Serologic and hexon phylogenetic analysis of ruminant adenoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to determine the antigenic relationship among ruminant adenoviruses and determine their phylogenetic relationship based on the deduced hexon gene amino acid sequence. Results of reciprocal cross-neutralization tests demonstrated antigenic relationships in either on...

  19. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  20. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  1. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  2. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  3. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  4. Microscale receiver operating characteristic analysis of micrometastasis recognition using activatable fluorescent probes indicates leukocyte imaging as a critical factor to enhance accuracy

    NASA Astrophysics Data System (ADS)

    Spring, Bryan Q.; Palanisami, Akilan; Hasan, Tayyaba

    2014-06-01

    Molecular-targeted probes are emerging with applications for optical biopsy of cancer. An underexplored potential clinical use of these probes is to monitor residual cancer micrometastases that escape cytoreductive surgery and chemotherapy. Here, we show that leukocytes, or white blood cells, residing in nontumor tissues-as well as those infiltrating micrometastatic lesions-uptake cancer cell-targeted, activatable immunoconjugates nonspecifically, which limits the accuracy and resolution of micrometastasis recognition using these probes. Receiver operating characteristic analysis of freshly excised tissues from a mouse model of peritoneal carcinomatosis suggests that dual-color imaging, adding an immunostain for leukocytes, offers promise for enabling accurate recognition of single cancer cells. Our results indicate that leukocyte identification improves micrometastasis recognition sensitivity and specificity from 92 to 93%-for multicellular metastases >20 to 30 μm in size-to 98 to 99.9% for resolving metastases as small as a single cell.

  5. Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo.

    PubMed

    Zhong, Yinan; Goltsche, Katharina; Cheng, Liang; Xie, Fang; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan; Haag, Rainer

    2016-04-01

    The therapeutic efficacy of nanoscale anticancer drug delivery systems is severely truncated by their low tumor-targetability and inefficient drug release at the target site. Here, we report the design and development of novel endosomal pH-activatable paclitaxel prodrug micelles based on hyaluronic acid-b-dendritic oligoglycerol (HA-dOG-PTX-PM) for active targeting and effective treatment of CD44-overexpressing human breast cancer xenografts in nude mice. HA-dOG-PTX-PM had a high drug content of 20.6 wt.% and an average diameter of 155 nm. The release of PTX was slow at pH 7.4 but greatly accelerated at endosomal pH. MTT assays, flow cytometry and confocal experiments showed that HA-dOG-PTX-PM possessed a high targetability and antitumor activity toward CD44 receptor overexpressing MCF-7 human breast cancer cells. The in vivo pharmacokinetics and biodistribution studies showed that HA-dOG-PTX-PM had a prolonged circulation time in the nude mice and a remarkably high accumulation in the MCF-7 tumor (6.19%ID/g at 12 h post injection). Interestingly, HA-dOG-PTX-PM could effectively treat mice bearing MCF-7 human breast tumor xenografts with little side effects, resulting in complete inhibition of tumor growth and a 100% survival rate over an experimental period of 55 days. These results indicate that hyaluronic acid-shelled acid-activatable PTX prodrug micelles have a great potential for targeted chemotherapy of CD44-positive cancers. PMID:26851390

  6. Immunization with adenovirus LIGHT-engineered dendritic cells induces potent T cell responses and therapeutic immunity in HBV transgenic mice.

    PubMed

    Jiang, Wenzheng; Chen, Ran; Kong, Xiaobo; Long, Fengying; Shi, Yaru

    2014-07-31

    LIGHT, a TNF superfamily member (TNFSF14), is a type II transmembrane protein expressed on activated T cells and immature dendritic cells (DCs). However, the expression of LIGHT on mature DCs is down-regulated. Recent studies demonstrated that LIGHT provides potent costimulatory activity for T cells, enhancing proliferation and the production of Th1 cytokines independently of the B7-CD28 pathway. Here, we evaluated the effectiveness of peptide-pulsed DC-mediated antiviral immunity in HBV transgenic mice and the immunoadjuvant effect of LIGHT. The bone marrow-derived DCs were modified in vitro with an adenovirus (Ad) vector expressing mouse LIGHT (Ad-LIGHT), the expression of costimulatory molecules was up-regulated and the secretion of cytokines IL-12 and IFN-γ increased. LIGHT-modified DCs enhanced allostimulation for T cells in mixed lymphocyte reaction (MLR). HBV peptide-pulsed DCs elicited HBV specific CD8+ T cell response and reduced the level of HBsAg and HBV DNA in sera of HBV transgenic mice. Importantly, LIGHT-modified DCs could induce stronger antiviral immunity. These results support the concept that genetic modification of DCs with a recombinant LIGHT adenovirus vector may be a useful strategy for antiviral immunotherapy. PMID:24951859

  7. 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

  8. 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.

  9. A Combinatory Strategy for Detection of Live CTCs Using Microfiltration and a New Telomerase-Selective Adenovirus.

    PubMed

    Ma, Yanchun; Hao, Sijie; Wang, Shuwen; Zhao, Yuanjun; Lim, Bora; Lei, Ming; Spector, David J; El-Deiry, Wafik S; Zheng, Si-Yang; Zhu, Jiyue

    2015-03-01

    Circulating tumor cells (CTC) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of patients with cancer. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared with the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of patients with breast and pancreatic cancer, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. PMID:25589497

  10. Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors

    PubMed Central

    Greish, Khaled; Frandsen, Jordan; Scharff, Stephanie; Gustafson, Joshua; Cappello, Joseph; Li, Daqing; O’Malley, Bert W.; Ghandehari, Hamidreza

    2010-01-01

    Background Adenoviral directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. Challenges with this approach however are transient gene expression and dissemination of viruses to distant organs. Methods We used recombinant silk-elastinlike protein copolymer (SELP) matrices for intratumoral delivery of adenoviruses containing both thymidine kinase-1, and luciferase genes in a nude mice model of JHU-022 head and neck tumor. Hydrogels made from two SELP analogues (47K and 815K) with similar silk to elastinlike block ratios but different block lengths were studied for intratumoral viral delivery. Tumor bearing mice were followed up for tumor progression and luciferase gene expression concomitantly for five weeks. Polymer’s safety was evaluated through body weight change, blood count, liver and kidney functions in addition to gross and microscopic histological examination. Results SELP 815K analogues efficiently controlled the duration and extent of transfection in tumors for up to 5 weeks with no detectable spread to the liver. About five-fold greater reduction in tumor volume was obtained with matrix-mediated delivery compared to intra-tumoral injection of adenoviruses in saline. SELP matrix proved safe in all injected mice compared to control group. Conclusion SELP- controlled gene delivery approach could potentially improve the anticancer activity of virus-mediated gene therapy while limiting viral spread to normal organs. PMID:20603862

  11. 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

  12. Functional Heterogeneity of Virions in Human Adenovirus Types 2 and 12

    PubMed Central

    Rainbow, Andrew J.; Mak, Stanley

    1970-01-01

    Purified preparations of adenovirus types 2 and 12 were used to infect KB cells at different input multiplicities. The resulting infected cultures were scored for inclusion body formation, production of infectious centers, and cloning efficiency. Both preparations were found to contain some defective particles capable of preventing a cell from cloning but unable to induce inclusion bodies or form plaques. The proportion of such defective particles in adenovirus 12 was about 10 times that in adenovirus 2. At high input multiplicities, the percentage of cells displaying an inclusion body was less than that predicted by the Poisson distribution and reached a maximum of 40 to 60% for adenovirus 2 and 12 to 15% for adenovirus 12. This reduction may be due to interference by large numbers of non-plaque-producing particles infecting each cell. The per cent of cells forming infectious centers was substantially greater for adenovirus 2 than for adenovirus 12 when compared at the same input plaque-forming units, reaching a maximum of 35 to 73% for adenovirus 2 and 5 to 10% for adenovirus 12. The low value for adenovirus 12 may be a result of the same interference phenomenon. Images PMID:4194167

  13. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

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

    PubMed Central

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

    2014-01-01

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

  15. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis.

    PubMed Central

    Babiss, L E; Ginsberg, H S

    1984-01-01

    To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or

  16. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis.

    PubMed

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

    2015-11-27

    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development. PMID:26403370

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

    PubMed Central

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

    1970-01-01

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

  18. A Promising Trigene Recombinant Human Adenovirus Vaccine Against Classical Swine Fever Virus.

    PubMed

    Li, Helin; Gao, Rui; Zhang, Yanming

    2016-05-01

    Classical swine fever (CSF) vaccine based on HAdV-5 had achieved an efficient protection in swine. Both classical swine fever virus (CSFV) E0 glycoprotein and E2 glycoprotein were the targets for neutralizing antibodies and related to immune protection against CSF. Interleukin-2 (IL2), as an adjuvant, also had been used in CSF vaccine research. In this study, coexpression of the CSFV E0, E2, and IL2 genes by HAdV-5 (rAdV-E0-E2-IL2) was constructed and immunized to evaluate its efficacy. Three expressed genes had been sequentially connected with foot-and-mouth disease virus 2A (FMDV 2A). The vaccine was administered by intramuscular inoculation to CSFV-free pigs (10(8) TCID50) twice at triweekly intervals. No adverse clinical signs were observed in any of the pigs after vaccination. The vaccine induced strong humoral and cellular responses that led to complete protection against clinical signs of lethal CSFV infection, viremia, and shedding of challenge virus. The rAdV-E0-E2-IL2 is a promising, efficient, and safe marker vaccine candidate against CSFV. PMID:26918463

  19. Construction of a fowl adenovirus recombinant to express avian metapneumovirus glycoprotein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian metapneumovirus (aMPV) is the cause of severe respiratory infection in turkeys. Despite detailed sequence analyses of most of the aMPV genes, very little is known about the role these proteins in viral virulence, pathogenesis, and immune response. Here, we report the construction of an avian a...

  20. [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yang, Yang; Wen, Bo; Tan, Wenjie

    2015-01-01

    To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine. PMID:25997323

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1986-01-01

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

  3. Identification of Adenoviruses in Specimens from High-Risk Pediatric Stem Cell Transplant Recipients and Controls▿

    PubMed Central

    Zheng, Xiaotian; Lu, Xiaoyan; Erdman, Dean D.; Anderson, Evan J.; Guzman-Cottrill, Judith A.; Kletzel, Morris; Katz, Ben Z.

    2008-01-01

    Adenovirus infection is an important cause of morbidity and mortality in stem cell transplant recipients. We report species and type-specific analysis from a prospective study of high-risk adenovirus infections following hematopoietic progenitor cell transplantation prior to, during, and after treatment with cidofovir, as well as species analysis of contemporaneously collected samples from control patients. Nine different adenovirus types representing all six recognized species were identified, and mixed infections were commonly found in this group of patients. PMID:17989198

  4. Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy.

    PubMed

    Kumar, Anuj; Das, Soma; Mullick, Ranajoy; Lahiri, Priyanka; Tatineni, Ranjitha; Goswami, Debashree; Bhat, Prasanna; Torresi, Joseph; Gowans, Eric James; Karande, Anjali Anoop; Das, Saumitra

    2016-02-17

    Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy. PMID:26700891

  5. First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

    PubMed

    Lima, Francisco Esmaile de Sales; Cibulski, Samuel Paulo; Elesbao, Felipe; Carnieli Junior, Pedro; Batista, Helena Beatriz de Carvalho Ruthner; Roehe, Paulo Michel; Franco, Ana Cláudia

    2013-10-01

    This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses. PMID:23828618

  6. Phylogenetic Analyses of Novel Squamate Adenovirus Sequences in Wild-Caught Anolis Lizards

    PubMed Central

    Ascher, Jill M.; Geneva, Anthony J.; Ng, Julienne; Wyatt, Jeffrey D.; Glor, Richard E.

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity. PMID:23593364

  7. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  8. Novel bat adenoviruses with an extremely large E3 gene.

    PubMed

    Tan, Bing; Yang, Xing-Lou; Ge, Xing-Yi; Peng, Cheng; Zhang, Yun-Zhi; Zhang, Li-Biao; Shi, Zheng-Li

    2016-07-01

    Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs. PMID:27032099

  9. Adenovirus type 3 pneumonia causing lung damage in childhood.

    PubMed Central

    Herbert, F. A.; Wilkinson, D.; Burchak, E.; Morgante, O.

    1977-01-01

    An outbreak of adenovirus type 3 infection occurred in a hospital in 19 North American Indian infants and young children who were being treated for unrelated problems. Pneumonia occurred in 14 and was usually severe, with persistent signs of airway obstruction. Eleven of the 14 were followed periodically and complete medical reviews were conducted 8 to 10 years later. Ten had abnormal chest radiographs, and bronchography revealed bronchiectasis and minor airways changes in seven. In three cases there was clear evidence that these changes were directly related to the adenovirus type 3 infection. Pulmonary function studies showed a combination of restrictive and obstructive changes with minimal hypoxemia in most. Despite the presence of a persistent productive cough all were able to carry on a relatively normal life. Images FIG. 1 FIG. 2 FIG. 3 PMID:189889

  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. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    NASA Astrophysics Data System (ADS)

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2016-01-01

    The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

  12. Effect of Relative Humidity on Dynamic Aerosols of Adenovirus 12

    PubMed Central

    Davis, Gary W.; Griesemer, Richard A.; Shadduck, John A.; Farrell, Robert L.

    1971-01-01

    Dynamic aerosols of adenovirus 12 were generated in the same Henderson apparatus under conditions of high, medium, and low relative humidity. High relative humidities resulted in more recovery of adenovirus 12 from aerosols and lungs of newborn Syrian hamsters. At 89, 51, and 32% relative humidity, the total infectious virus recovered from a 20-min aerosol was 106.7, 106.0, and 104.3 TCD50, respectively. Hamsters exposed to these 20-min aerosols retained measured lung doses of 103.0, 102.4, and 101.0 TCD50, respectively. The measured retained lung doses were compared to calculated inhaled lung doses based on both total virus aerosolized and total virus recovery from the aerosols. PMID:4930277

  13. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  14. Oncolytic adenovirus-mediated therapy for prostate cancer.

    PubMed

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen-androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

  15. Oncolytic adenovirus-mediated therapy for prostate cancer

    PubMed Central

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

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

    SciTech Connect

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

    2006-09-30

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

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

    PubMed

    Singh, Ravi; Tian, Bowen; Kostarelos, Kostas

    2008-09-01

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

  18. Immunity against heterosubtypic influenza virus induced by adenovirus and MVA expressing nucleoprotein and matrix protein-1.

    PubMed

    Lambe, Teresa; Carey, John B; Li, Yuanyuan; Spencer, Alexandra J; van Laarhoven, Arjan; Mullarkey, Caitlin E; Vrdoljak, Anto; Moore, Anne C; Gilbert, Sarah C

    2013-01-01

    Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8(+) T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8(+) T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants. PMID:23485942

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed

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

    2016-04-01

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

  1. Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

    PubMed Central

    Wang, Xu-Shan; Khuntirat, Benjawan; Qing, Keyun; Ponnazhagan, Selvarangan; Kube, Dagmar M.; Zhou, Shangzhen; Dwarki, Varavani J.; Srivastava, Arun

    1998-01-01

    The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence

  2. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  3. 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

  4. An outbreak of lethal adenovirus infection among different otariid species.

    PubMed

    Inoshima, Yasuo; Murakami, Tomoaki; Ishiguro, Naotaka; Hasegawa, Kazuhiro; Kasamatsu, Masahiko

    2013-08-30

    An outbreak of fatal fulminant hepatitis at a Japanese aquarium involved 3 otariids: a California sea lion (Zalophus californianus), a South African fur seal (Arctocephalus pusillus) and a South American sea lion (Otaria flavescens). In a span of about a week in February 2012, 3 otariids showed diarrhea and were acutely low-spirited; subsequently, all three animals died within a period of 3 days. Markedly increased aspartate amino transferase and alanine amino transferase activities were observed. Necrotic hepatitis and eosinophilic intranuclear inclusion bodies in liver hepatocytes and intestinal epithelial cells were observed in the South American sea lion on histological examination. Otarine adenovirus DNA was detected from the livers of all three animals by polymerase chain reaction and determination of the sequences showed that all were identical. These results suggest that a single otarine adenovirus strain may have been the etiological agent of this outbreak of fatal fulminant hepatitis among the different otariid species, and it may be a lethal threat to wild and captive otariids. This is the first evidence of an outbreak of lethal adenovirus infection among different otariid species. PMID:23643878

  5. History of the restoration of adenovirus type 4 and type 7 vaccine, live oral (Adenovirus Vaccine) in the context of the Department of Defense acquisition system.

    PubMed

    Hoke, Charles H; Snyder, Clifford E

    2013-03-15

    Respiratory pathogens cause morbidity and mortality in US military basic trainees. Following the influenza pandemic of 1918, and stimulated by WWII, the need to protect military personnel against epidemic respiratory disease was evident. Over several decades, the US military elucidated etiologies of acute respiratory diseases and invented and deployed vaccines to prevent disease caused by influenza, meningococcus, and adenoviruses. In 1994, the Adenovirus Vaccine manufacturer stopped its production. By 1999, supplies were exhausted and adenovirus-associated disease, especially serotype 4-associated febrile respiratory illness, returned to basic training installations. Advisory bodies persuaded Department of Defense leaders to initiate restoration of Adenovirus Vaccine. In 2011, after 10 years of effort by government and contractor personnel and at a cost of about $100 million, the Adenovirus Vaccine was restored to use at all military basic training installations. Disease and adenovirus serotype 4 isolation rates have fallen dramatically since vaccinations resumed in October 2011 and remain very low. Mindful of the adage that "The more successful a vaccine is, the more quickly the need for it will be forgotten.", sustainment of the supply of the Adenovirus Vaccine may be a challenge, and careful management will be required for such sustainment. PMID:23291475

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

    PubMed

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

    2005-04-01

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

  7. Detection of Known and Novel Adenoviruses in Cattle Wastes via Broad-Spectrum Primers ▿

    PubMed Central

    Sibley, Samuel D.; Goldberg, Tony L.; Pedersen, Joel A.

    2011-01-01

    The critical assessment of bovine adenoviruses (BAdV) as indicators of environmental fecal contamination requires improved knowledge of their prevalence, shedding dynamics, and genetic diversity. We examined DNA extracted from bovine and other animal waste samples collected in Wisconsin for atadenoviruses and mastadenoviruses using novel, broad-spectrum PCR primer sets. BAdV were detected in 13% of cattle fecal samples, 90% of cattle urine samples, and 100% of cattle manure samples; 44 percent of BAdV-positive samples contained both Atadenovirus and Mastadenovirus DNA. Additionally, BAdV were detected in soil, runoff water from a cattle feedlot, and residential well water. Overall, we detected 8 of 11 prototype BAdV, plus bovine, rabbit, and porcine mastadenoviruses that diverged significantly from previously reported genotypes. The prevalence of BAdV shedding by cattle supports targeting AdV broadly as indicators of the presence of fecal contamination in aqueous environments. Conversely, several factors complicate the use of AdV for fecal source attribution. Animal AdV infecting a given livestock host were not monophyletic, recombination among livestock mastadenoviruses was detected, and the genetic diversity of animal AdV is still underreported. These caveats highlight the need for continuing genetic surveillance for animal AdV and for supporting data when BAdV detection is invoked for fecal source attribution in environmental samples. To our knowledge, this is the first study to report natural BAdV excretion in urine, BAdV detection in groundwater, and recombination in AdV of livestock origin. PMID:21622778

  8. The Evaluation of Polyhexamethylene Biguanide (PHMB) as a Disinfectant for Adenovirus

    PubMed Central

    Romanowski, Eric G.; Yates, Kathleen A.; O’Connor, Katherine E.; Mah, Francis S.; Shanks, Robert M. Q.; Kowalski, Regis P.

    2013-01-01

    Purpose Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs. The current study determined whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs. Methods The direct disinfecting activity of PHMB was determined in triplicate assays by incubating nine human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with 50 and 0 PPM (µg/ml) of PHMB for 24 hours at room temperature, to simulate swimming pool temperatures, or 40°C, to simulate hot tub temperatures. Plaque assays determined adenovirus titers after incubation. Titers were Log10 converted and mean ± standard deviation Log10 reductions from controls were calculated. Virucidal (greater than 99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested. Results At room temperature, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for all adenovirus types tested. At 40°C, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for two adenovirus types and greater than 1 Log10, but less than 3 Log10, for seven of nine adenovirus types. Conclusions 50 PPM of PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Clinical Relevance Recreational water maintained and sanitized with PHMB has the potential to serve as a vector for the transmission of ocular adenovirus infections. PMID:23450376

  9. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  10. Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins.

    PubMed Central

    Harter, M L; Lewis, J B; Anderson, C W

    1979-01-01

    The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon. Images PMID:513195

  11. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    PubMed

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  12. 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...

  13. 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.

  14. 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

  15. Adenovirus Type 7 Pneumonia in Children Who Died from Measles-Associated Pneumonia, Hanoi, Vietnam, 2014

    PubMed Central

    Hai, Le Thanh; Thach, Hoang Ngoc; Tuan, Ta Anh; Nam, Dao Huu; Dien, Tran Minh; Sato, Yuko; Kumasaka, Toshio; Suzuki, Tadaki; Hanaoka, Nozomu; Fujimoto, Tsuguto; Katano, Harutaka; Hasegawa, Hideki; Kawachi, Shoji

    2016-01-01

    During a 2014 measles outbreak in Vietnam, postmortem pathologic examination of hospitalized children who died showed that adenovirus type 7 pneumonia was a contributory cause of death in children with measles-associated immune suppression. Adenovirus type 7 pneumonia should be recognized as a major cause of secondary infection after measles. PMID:26926035

  16. Adenovirus-based vaccines against avian-origin H5N1 influenza viruses.

    PubMed

    He, Biao; Zheng, Bo-jian; Wang, Qian; Du, Lanying; Jiang, Shibo; Lu, Lu

    2015-02-01

    Since 1997, human infection with avian H5N1, having about 60% mortality, has posed a threat to public health. In this review, we describe the epidemiology of H5N1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based H5N1 systemic and mucosal vaccines. PMID:25479556

  17. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO ENTERIC ADENOVIRUS TYPES 40 AND 41

    EPA Science Inventory

    The authors have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad4...

  18. 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...

  19. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

    PubMed Central

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H. J.; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  20. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

    PubMed

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H J; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  1. Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy.

    PubMed

    Thapa, Pritam; Li, Mengjie; Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; Sun, Yajing; Woo, Sukyung; You, Youngjae

    2016-04-14

    Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy. PMID:26974508

  2. The effect of "on/off" molecular switching on the photophysical and photochemical properties of axially calixarene substituted activatable silicon(iv)phthalocyanine photosensitizers.

    PubMed

    Güngör, Ömer; Altınbaş Özpınar, Gül; Durmuş, Mahmut; Ahsen, Vefa

    2016-05-01

    Silicon(iv) phthalocyanines ( and ) bearing two calixarene groups as axial ligands were synthesized. Surprisingly, both phthalocyanines were obtained as two different isomers ( and ) depending on the distance between calixarene benzene groups and the phthalocyanine ring. DFT and TD-DFT computations were performed to model plausible structures of these isomers and to simulate electronic absorption spectra. These isomers converted into each other depending on the polarity of the used solvent, temperature and light irradiation. The photophysical and photochemical properties of each isomer were investigated in dimethylsulfoxide (DMSO) for the determination of photodynamic therapy (PDT) activities of these compounds. The more blue-shifted isomers ( and ) showed higher fluorescence quantum yields and singlet oxygen generation compared to more red-shifted counterparts ( and ). This behavior is extremely important for developing activatable photosensitizers for cancer treatment by PDT. Although these photosensitizers produce lower singlet oxygen in normal cells, they produce higher singlet oxygen (six times higher for ) in cancer cells since these photosensitizers converted to more blue-shifted isomers by using light irradiation. PMID:27052992

  3. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  4. Longitudinal Requirement for CD4+ T Cell Help for Adenovirus Vector–Elicited CD8+ T Cell Responses

    PubMed Central

    Provine, Nicholas M.; Larocca, Rafael A.; Penaloza-MacMaster, Pablo; Borducchi, Erica N.; McNally, Anna; Parenteau, Lily R.; Kaufman, David R.

    2014-01-01

    Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8+ T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8+ T cell responses by Ad vector immunization is longitudinally dependent on CD4+ T cell help for a prolonged period. Depletion of CD4+ T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8+ T cells, decreased T-bet and eomesodermin expression, impaired KLRG1+ effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8+ T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4+ T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8+ T cells. These data demonstrate a prolonged temporal requirement for CD4+ T cell help for vaccine-elicited CD8+ T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8+ T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4+ T cell immune deficiencies. PMID:24778441

  5. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  6. 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

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

    PubMed Central

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

    2014-01-01

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

  8. Proteins encoded near the adenovirus late messenger RNA leader segments

    SciTech Connect

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

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

    PubMed

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

    2014-12-01

    A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing. PMID:25632689

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

    PubMed

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

    2016-08-01

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

  11. Dendritic cells serve as a “Trojan horse” for oncolytic adenovirus delivery in the treatment of mouse prostate cancer

    PubMed Central

    Li, Zhao-lun; Liang, Xuan; Li, He-cheng; Wang, Zi-ming; Chong, Tie

    2016-01-01

    Aim: Adenovirus-mediated gene therapy is a novel therapeutic approach for the treatment of cancer, in which replication of the virus itself is the anticancer method. However, the success of this novel therapy is limited due to inefficient delivery of the virus to the target sites. In this study, we used dendritic cells (DCs) as carriers for conditionally replicating adenoviruses (CRAds) in targeting prostate carcinoma (PCa). Methods: Four types of CRAds, including Ad-PC (without PCa-specific promoter and a recombinant human tumor necrosis factor, rmhTNF, sequence), Ad-PC-rmhTNF (without PCa-specific promoter), Ad-PPC-NCS (without an rmhTNF sequence) and Ad-PPC-rmhTNF, were constructed. The androgen-insensitive mouse PCa RM-1 cells were co-cultured with CRAd-loading DCs, and the viability of RM-1 cells was examined using MTT assay. The in vivo effects of CRAd-loading DCs on PCa were evaluated in RM-1 xenograft mouse model. Results: Two PCa-specific CRAds (Ad-PPC-NCS, Ad-PPC-rmhTNF) exhibited more potent suppression on the viability of RM-1 cells in vitro than the PCa-non-specific CRAds (Ad-PC, Ad-PC-rmhTNF). In PCa-bearing mice, intravenous injection of the PCa-specific CRAd-loading DCs significantly inhibited the growth of xenografted tumors, extended the survival time, and induced T-cell activation. Additionally, the rmhTNF-containing CRAds exhibited greater tumor killing ability than CRAds without rmhTNF. Conclusion: DCs may be an effective vector for the delivery of CRAds in the treatment of PCa. PMID:27345628

  12. Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene

    SciTech Connect

    Moran, B.; Zerler, B.

    1988-04-01

    Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, the authors constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products. This deletion did not impede the ability of the E1A products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.

  13. Recombinant modified vaccinia virus Ankara-based malaria vaccines.

    PubMed

    Sebastian, Sarah; Gilbert, Sarah C

    2016-01-01

    A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years. PMID:26511884

  14. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  15. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    PubMed

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

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

    PubMed

    Gordon, J W

    2001-04-01

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

  17. 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

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

    USGS Publications Warehouse

    Counihan, Katrina; Skerratt, Lee; Franson, J. Christian; Hollmen, Tuula E.

    2015-01-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

  19. Neonatal Infection with Species C Adenoviruses Confirmed in Viable Cord Blood Lymphocytes

    PubMed Central

    Ornelles, David A.; Gooding, Linda R.; Garnett-Benson, C.

    2015-01-01

    Credible but conflicting reports address the frequency of prenatal infection by species C adenovirus. This question is important because these viruses persist in lymphoid cells and suppress double-stranded DNA-break repair. Consequently, prenatal adenovirus infections may generate the aberrant clones of lymphocytes that precede development of childhood acute lymphoblastic leukemia (ALL). The present study was designed to overcome technical limitations of prior work by processing cord blood lymphocytes within a day of collection, and by analyzing sufficient numbers of lymphocytes to detect adenovirus-containing cells at the lower limits determined by our previous studies of tonsil lymphocytes. By this approach, adenoviral DNA was identified in 19 of 517 (3.7%) samples, providing definitive evidence for the occurrence of prenatal infection with species C adenoviruses in a significant fraction of neonates predominantly of African American and Hispanic ancestry. Cord blood samples were also tested for the presence of the ETV6-RUNX1 translocation, the most common genetic abnormality in childhood ALL. Using a nested PCR assay, the ETV6-RUNX1 transcript was detected in four of 196 adenovirus-negative samples and one of 14 adenovirus-positive cord blood samples. These findings indicate that this method will be suitable for determining concordance between adenovirus infection and the leukemia-associated translocations in newborns. PMID:25764068

  20. Neonatal infection with species C adenoviruses confirmed in viable cord blood lymphocytes.

    PubMed

    Ornelles, David A; Gooding, Linda R; Garnett-Benson, C

    2015-01-01

    Credible but conflicting reports address the frequency of prenatal infection by species C adenovirus. This question is important because these viruses persist in lymphoid cells and suppress double-stranded DNA-break repair. Consequently, prenatal adenovirus infections may generate the aberrant clones of lymphocytes that precede development of childhood acute lymphoblastic leukemia (ALL). The present study was designed to overcome technical limitations of prior work by processing cord blood lymphocytes within a day of collection, and by analyzing sufficient numbers of lymphocytes to detect adenovirus-containing cells at the lower limits determined by our previous studies of tonsil lymphocytes. By this approach, adenoviral DNA was identified in 19 of 517 (3.7%) samples, providing definitive evidence for the occurrence of prenatal infection with species C adenoviruses in a significant fraction of neonates predominantly of African American and Hispanic ancestry. Cord blood samples were also tested for the presence of the ETV6-RUNX1 translocation, the most common genetic abnormality in childhood ALL. Using a nested PCR assay, the ETV6-RUNX1 transcript was detected in four of 196 adenovirus-negative samples and one of 14 adenovirus-positive cord blood samples. These findings indicate that this method will be suitable for determining concordance between adenovirus infection and the leukemia-associated translocations in newborns. PMID:25764068

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

    PubMed

    Walters, R; Welsh, M

    1999-11-01

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

  2. Application of the polymerase chain reaction to detect fowl adenoviruses.

    PubMed Central

    Jiang, P; Ojkic, D; Tuboly, T; Huber, P; Nagy, E

    1999-01-01

    The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative. Images Figure 2. Figure 3. Figure 4. PMID:10369570

  3. Purification of a native membrane-associated adenovirus tumor antigen.

    PubMed Central

    Persson, H; Katze, M G; Philipson, L

    1982-01-01

    A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro. Images PMID:7097863

  4. 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

  5. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

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

    2015-08-15

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

  6. Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses

    PubMed Central

    Báez-Astúa, Andrés; Herráez-Hernández, Elsa; Garbi, Natalio; Pasolli, Hilda A.; Juárez, Victoria; zur Hausen, Harald; Cid-Arregui, Angel

    2005-01-01

    Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (106 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8+-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein. PMID:16188983

  7. Molecular characterization of adenovirus circulating in Central and South America during the 2006–2008 period

    PubMed Central

    García, Josefina; Sovero, Merly; Laguna‐Torres, Victor Alberto; Gomez, Jorge; Chicaiza, Wilson; Barrantes, Melvin; Sanchez, Felix; Jimenez, Mirna; Comach, Guillermo; De Rivera, Ivette L.; Agudo, Roberto; Arango, Ana E.; Barboza, Alma; Aguayo, Nicolas; Kochel, Tadeusz J.

    2009-01-01

    Background  Human Adenoviruses are recognized pathogens, causing a broad spectrum of diseases. Serotype identification is critical for epidemiological surveillance, detection of new strains and understanding of HAdvs pathogenesis. Little data is available about HAdvs subtypes in Latin America. Methods  In this study, we have molecularly characterized 213 adenoviruses collected from ILI presenting patients, during 2006‐08, in Central and South America. Results  Our results indicate that 161(76%) adenoviruses belong to subgroup C, 45 (21%) to subgroup B and 7 (3%) to subtype E4. PMID:19903214

  8. Molecular detection of two adenoviruses associated with disease in Australian lizards.

    PubMed

    Hyndman, T; Shilton, C M

    2011-06-01

    We give the first published description of the pathology and molecular findings associated with adenovirus infection in lizards in Australia. A central netted dragon (Ctenophorus nuchalis) exhibited severe necrotising hepatitis with abundant intranuclear inclusion bodies within hepatocytes and rarely within intestinal epithelial cells. Polymerase chain reaction (PCR) using pooled tissues yielded an amplicon that shared strong nucleotide identity with an agamid adenovirus (EU914203). PCR on the liver of a bearded dragon (Pogona minor minor) with illthrift, coccidiosis, nematodiasis and hepatic lipidosis yielded an amplicon with strong nucleotide identity to a helodermatid adenovirus (EU914207). PMID:21595645

  9. In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes.

    PubMed

    Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid

    2014-07-28

    Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression. PMID

  10. Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

    PubMed

    Simard, Bryan; Mironov, Gleb G; Tomanek, Boguslaw; van Veggel, Frank C J M; Abulrob, Abedelnasser

    2016-06-01

    Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27282138

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

    PubMed Central

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

    2011-01-01

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

  12. Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization▿

    PubMed Central

    Cheng, Cheng; Gall, Jason G. D.; Nason, Martha; King, C. Richter; Koup, Richard A.; Roederer, Mario; McElrath, M. Juliana; Morgan, Cecilia A.; Churchyard, Gavin; Baden, Lindsey R.; Duerr, Ann C.; Keefer, Michael C.; Graham, Barney S.; Nabel, Gary J.

    2010-01-01

    A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors. PMID:19846512

  13. Epithelial Junction Opener Improves Oncolytic Adenovirus Therapy in Mouse Tumor Models.

    PubMed

    Yumul, Roma; Richter, Maximilian; Lu, Zhuo-Zhuang; Saydaminova, Kamola; Wang, Hongjie; Wang, Chung-Huei Katherine; Carter, Darrick; Lieber, André

    2016-04-01

    A central resistance mechanism in solid tumors is the maintenance of epithelial junctions between malignant cells that prevent drug penetration into the tumor. Human adenoviruses (Ads) have evolved mechanisms to breach epithelial barriers. For example, during Ad serotype 3 (Ad3) infection of epithelial tumor cells, massive amounts of subviral penton-dodecahedral particles (PtDd) are produced and released from infected cells to trigger the transient opening of epithelial junctions, thus facilitating lateral virus spread. We show here that an Ad3 mutant that is disabled for PtDd production is significantly less effective in killing of epithelial human xenograft tumors than the wild-type Ad3 virus. Intratumoral spread and therapeutic effect of the Ad3 mutant was enhanced by co-administration of a small recombinant protein (JO; produced in Escherichia coli) that incorporated the minimal junction opening domains of PtDd. We then demonstrated that co-administration of JO with replication-competent Ads that do not produce PtDd (Ad5, Ad35) resulted in greater attenuation of tumor growth than virus injection alone. Furthermore, we genetically modified a conditionally replicating Ad5-based oncolytic Ad (Ad5Δ24) to express a secreted form of JO upon replication in tumor cells. The JO-expressing virus had a significantly greater antitumor effect than the unmodified AdΔ24 version. Our findings indicate that epithelial junctions limit the efficacy of oncolytic Ads and that this problem can be address by co-injection or expression of JO. JO has also the potential for improving cancer therapy with other types of oncolytic viruses. PMID:26993072

  14. Delineation of Interfaces on Human Alpha-Defensins Critical for Human Adenovirus and Human Papillomavirus Inhibition

    PubMed Central

    Wiens, Mayim E.; Lu, Wuyuan; Smith, Jason G.

    2014-01-01

    Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity. PMID:25188351

  15. Structural and Functional Studies on the Interaction of Adenovirus Fiber Knobs and Desmoglein 2

    PubMed Central

    Wang, Hongjie; Yumul, Roma; Cao, Hua; Ran, Liang; Fan, Xiaolong; Richter, Maximilian; Epstein, Forrest; Gralow, Julie; Zubieta, Chloe

    2013-01-01

    Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. Our findings have practical relevance for cancer therapy. We have recently reported that an Ad3 fiber knob-containing recombinant protein (JO-1) is able to trigger opening of junctions between epithelial cancer cells which, in turn, greatly improved the intratumoral penetration and efficacy of therapeutic agents (I. Beyer, et al., Clin. Cancer Res. 18:3340–3351, 2012; I. Beyer, et al., Cancer Res. 71:7080–7090, 2011). Here, we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models. PMID:23946456

  16. Potent and long-term antiangiogenic efficacy mediated by FP3-expressing oncolytic adenovirus.

    PubMed

    Choi, Il-Kyu; Shin, Hyewon; Oh, Eonju; Yoo, Ji Young; Hwang, June Kyu; Shin, Kyungsub; Yu, De-Chao; Yun, Chae-Ok

    2015-11-01

    Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases. PMID:25944623

  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. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  19. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  20. 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

  1. Molecular Detection of Adenoviruses, Rhabdoviruses, and Paramyxoviruses in Bats from Kenya

    PubMed Central

    Conrardy, Christina; Tao, Ying; Kuzmin, Ivan V.; Niezgoda, Michael; Agwanda, Bernard; Breiman, Robert F.; Anderson, Larry J.; Rupprecht, Charles E.; Tong, Suxiang

    2014-01-01

    We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus. PMID:24865685

  2. 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...

  3. Molecular detection of adenoviruses, rhabdoviruses, and paramyxoviruses in bats from Kenya.

    PubMed

    Conrardy, Christina; Tao, Ying; Kuzmin, Ivan V; Niezgoda, Michael; Agwanda, Bernard; Breiman, Robert F; Anderson, Larry J; Rupprecht, Charles E; Tong, Suxiang

    2014-08-01

    We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus. PMID:24865685

  4. 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.

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2008-05-19

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

  7. 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

  8. Probing intra-cellular drug release event using activatable (OFF/ON) CdS:Mn/ZnS quantum dots (Qdots): spectroscopic studies to investigate interaction of Qdots with quencher

    NASA Astrophysics Data System (ADS)

    Tharkur, Jeremy; Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Cantarero, Karishma; Maity, Niharika; Rifai, Sara; Doshi, Mona; Gesquiere, Andre J.; Santra, Swadeshmukul

    2013-02-01

    In recent years, activatable Quantum Dots (AQdots) are gaining popularity in a number of chemical and biological sensing applications. A basic design of AQdot probes involves a suitable quencher which is capable of altering optical properties of the Qdots. In our previous studies we have shown that CdS:Mn/ZnS fluorescence can be effectively quenched using small molecule quenchers (such as dopamine, chemotherapeutic drug) as well as iron oxide nanoparticle via electron/energy transfer process. We have also shown that the quenched Qdot fluorescence can be restored when the Qdots are separated from the quencher. Using Qdot based activatable probes, we detected intracellular drug release event. Qdot fluorescence was restored upon interaction with the intracellular glutathione (GSH). In this paper, we report a GSH induced quenching of water-soluble N-Acetyl Cysteine (NAC) surface-conjugated Cds:Mn/ZnS Qdots. Quenching of NAC-Qdots was due to aggregation of Qdots in solution. This aggregation induced fluorescence quenching phenomenon resembles with the self-quenching phenomenon of traditional organic fluorescence dyes at high concentrations. UV-VIS and fluorescence emission spectroscopy data support the interaction and binding of GSH with the NAC-Qdots. Increase in particle size due to GSH induced aggregation of NAC-Qdots was confirmed by the Dynamic Light Scattering (DLS) data.

  9. Mesangial Localization of Immune Complexes in Experimental Canine Adenovirus Glomerulonephritis

    PubMed Central

    Wright, N. G.; Morrison, W. I.; Thompson, H.; Cornwell, H. J. C.

    1974-01-01

    Each of a group of 14 dogs was infected experimentally by an intravenous dose of canine adenovirus calculated to allow survival until the initial stages of antibody production; the kidneys of infected dogs were examined during the period of 4-14 days after administration of virus. Proliferative glomerulonephritis with localization of IgG, C3 and viral antigen in mesangial regions was demonstrated. With the electron microscope, electron dense deposits were found scattered throughout the mesangium. There was proliferation of mesangial cells, infiltration into the glomerular tuft of polymorphonuclear leucocytes and, in some cases, focal glomerular necrosis with intracapsular and tubular haemorrhage. By means of an indirect immunofluorescence test, anti-viral antibody was detected in kidney eluates; anti-kidney antibody was not present. ImagesFigs. 5-8Figs. 9-10Figs. 1-4 PMID:4375485

  10. Purification of adenovirus hexon by high performance liquid chromatography.

    PubMed

    Siegel, S A; Hutchins, J E; Witt, D J

    1987-09-01

    Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology. PMID:3680460

  11. Adenovirus receptors and their implications in gene delivery

    PubMed Central

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

    2010-01-01

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

  12. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

  13. Intermediates in the Synthesis of Type 2 Adenovirus Deoxyribonucleic Acid

    PubMed Central

    Horwitz, Marshall S.

    1971-01-01

    Intermediates in the synthesis of adenovirus type 2 deoxyribonucleic acid (DNA) were studied in HeLa cells. Pieces of DNA smaller than the viral genome were demonstrated after labeling with 3H-thymidine for 10 to 240 sec. Intermediates as small as the Okazaki fragments (8 to 10S) do not predominate at any of the above times. No detectable addition of nucleotides to parental genome could be shown, nor was there any breakdown of recently synthesized viral DNA. The DNA intermediates were of viral origin for they hybridized to viral DNA and were made at a stage of the cell cycle (G2) when host DNA is not synthesized. PMID:5132696

  14. Oncolytic adenoviruses: A thorny path to glioma cure

    PubMed Central

    Ulasov, I.V.; Borovjagin, A.V.; Schroeder, B.A.; Baryshnikov, A.Y.

    2014-01-01

    Glioblastoma Multiforme (GBM) is a rapidly progressing brain tumor. Despite the relatively low percentage of cancer patients with glioma diagnoses, recent statistics indicate that the number of glioma patients may have increased over the past decade. Current therapeutic options for glioma patients include tumor resection, chemotherapy, and concomitant radiation therapy with an average survival of approximately 16 months. The rapid progression of gliomas has spurred the development of novel treatment options, such as cancer gene therapy and oncolytic virotherapy. Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach. Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment. PMID:25685829

  15. Partial characterization of new adenoviruses found in lizards.

    PubMed

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

    2014-06-01

    In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species. PMID:25000689

  16. 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

  17. Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre

    SciTech Connect

    El Bakkouri, Majida; Seiradake, Elena; Cusack, Stephen; Ruigrok, Rob W.H. Schoehn, Guy

    2008-08-15

    There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.

  18. 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

  19. Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells

    SciTech Connect

    Richardson, W.D.; Anderson, C.W.

    1984-08-01

    Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 M/sub r/ virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus. 26 references, 2 figures, 1 table.

  20. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.

    PubMed

    Walters, R W; Grunst, T; Bergelson, J M; Finberg, R W; Welsh, M J; Zabner, J

    1999-04-01

    Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I alpha-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency. PMID:10187807

  1. [Anti-adenovirus activity of a substance and medical form of ribamydil in cell culture].

    PubMed

    Nosach, L N; Diachenko, N S; Zhovnovataia, V L

    2009-01-01

    The inhibiting effect of ribamydil on adenovirus reproduction was studied under the determination of the number of cells with virus- induced DNA-containing intranucleus inclusion bodies and hexone antigen, the synthesis of adenovirus proteins and the infection virus by t he investigation. EC50 of ribamydil substance is 4-8 microg/ml, but complete suppression of adenovirus genome expression was found when adding ribamydil after the virus adsorption, in concentrations of 125-500 microg/ml. The original effect of ribamydil on the expression of adenovirus genome was found under its effect in concentration of 31 microg/ml. Intranucleus virus-induced inclusion bodies of the early type only were found under these conditions. Synthesis of the structural virus polypeptides, including hexone polypeptide (II) and non-structural polypeptide 100K, taking part in hexone trimerization, proceed intensively but without formation of immunologically active hexone. The inhibiting effect of officinal form of ribamydil was less expressed as compared with the substance (EC50: 62 microg/ml). The work results prove that the therapeutic effect of ribamydil (ribavirin) under treatment of adenovirus infections may be achieved in case when it is used in a dose excluding the expression of the adenovirus genome. PMID:20458939

  2. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

    PubMed

    Lyons, Mark; Onion, David; Green, Nicky K; Aslan, Kriss; Rajaratnam, Ratna; Bazan-Peregrino, Miriam; Phipps, Sue; Hale, Sarah; Mautner, Vivien; Seymour, Leonard W; Fisher, Kerry D

    2006-07-01

    Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access. PMID:16580883

  3. Use of cidofovir in pediatric patients with adenovirus infection

    PubMed Central

    Ganapathi, Lakshmi; Arnold, Alana; Jones, Sarah; Patterson, Al; Graham, Dionne; Harper, Marvin; Levy, Ofer

    2016-01-01

    Background: Adenoviruses contribute to morbidity and mortality among immunocompromised pediatric patients including stem cell and solid organ transplant recipients. Cidofovir (CDV), an antiviral compound approved by the FDA in 1996, is used for treatment of adenoviral (ADV) infections in immunocompromised patients despite concern of potential nephrotoxicity.   Methods: We conducted a retrospective 5-year review at Boston Children’s Hospital of 16 patients (mean age = 6.5 years) receiving 19 courses of CDV. During therapy all pertinent data elements were reviewed to characterize potential response to therapy and incidence of renal dysfunction.   Results: Of the 19 CDV courses prescribed, 16 courses (84%) were in patients who had a positive blood ADV Polymerase chain reaction (PCR) alone or in combination with positive ADV PCR/ Direct Immunofluorescence Assay (DFA) at another site. Respiratory symptoms with or without pneumonia were the most common presentation (10/19, 53%). In the majority of blood positive courses (10/16, 63%), viral clearance was also accompanied by clinical response. This was not the case in four courses where patients expired despite viral clearance, including one in which death was directly attributable to adenovirus. There was reversible renal dysfunction observed during the use of CDV. Conclusions:  CDV appeared safe and reasonably tolerated for treatment of ADV in this pediatric population and was associated with viral response and clinical improvement in the majority of patients but reversible renal dysfunction was a side effect. Further studies of the efficacy of CDV for immunocompromised children with ADV infection are warranted. PMID:27239277

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

    PubMed

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

    2010-04-01

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

  5. Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells.

    PubMed

    Durvasula, Kiranmai; Thulé, Peter M; Sambanis, Athanassios

    2012-04-01

    One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies. PMID:22094821

  6. Combinatorial Insulin Secretion Dynamics of Recombinant Hepatic and Enteroendocrine Cells

    PubMed Central

    Durvasula, Kiranmai; Thulé, Peter M.; Sambanis, Athanassios

    2012-01-01

    One of the more promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with one of three adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards pre-clinical studies. PMID:22094821

  7. Human adenovirus type 8 epidemic keratoconjunctivitis with large corneal epithelial full-layer detachment: an endemic outbreak with uncommon manifestations

    PubMed Central

    Lee, Yueh-Chang; Chen, Nancy; Huang, I-Tsong; Yang, Hui-Hua; Huang, Chin-Te; Chen, Li-Kuang; Sheu, Min-Muh

    2015-01-01

    Epidemic viral conjunctivitis is a highly contagious disease that is encountered year-round. The causative agents are mainly adenoviruses and enteroviruses. It occurs most commonly upon infection with subgroup D adenoviruses of types 8, 19, or 37. For common corneal involvement of human adenovirus type 8 epidemic keratoconjunctivitis, full-layer epithelial detachment is rarely seen. Herein, we report three cases of epidemic keratoconjunctivitis during an outbreak which manifested as large corneal epithelial full-layer detachment within a few days. The lesions healed without severe sequelae under proper treatment. The unique manifestation of this outbreak may indicate the evolution of human adenovirus type 8. PMID:26060391

  8. 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 c