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Sample records for recombinant adenovirus boosting

  1. Activated recombinant adenovirus proteinases

    SciTech Connect

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

    PubMed

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

    2010-09-15

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

  4. 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

  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. Co-factor activated recombinant adenovirus proteinases

    SciTech Connect

    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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Transductional targeting with recombinant adenovirus vectors.

    PubMed

    Legrand, Valerie; Leissner, Philippe; Winter, Arend; Mehtali, Majid; Lusky, Monika

    2002-09-01

    Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed. PMID:12189719

  12. Coacervate microspheres as carriers of recombinant adenoviruses.

    PubMed

    Kalyanasundaram, S; Feinstein, S; Nicholson, J P; Leong, K W; Garver, R I

    1999-01-01

    The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. A novel and simple method for construction of recombinant adenoviruses.

    PubMed

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin

    2006-07-19

    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

  18. [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

  19. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Construction and identification of recombinant adenovirus carrying human TIMP-1shRNA gene.

    PubMed

    Sun, Y L; Xie, H; Lin, H L; Feng, Q; Liu, Y

    2015-01-16

    The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.

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

    PubMed Central

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

    2015-01-01

    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

  4. 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

    2015-12-29

    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).

  5. 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

  6. Vaccination Using Recombinants Influenza and Adenoviruses Encoding Amastigote Surface Protein-2 Are Highly Effective on Protection against Trypanosoma cruzi Infection

    PubMed Central

    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

  7. Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

    PubMed

    Xia, Qiu; Geng, Fei; Zhang, Fang-Fang; Liu, Chen-Lu; Xu, Ping; Lu, Zhen-Zhen; Zhang, Hai-Hong; Kong, Wei; Yu, Xiang-Hui

    2016-08-31

    Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines. PMID:27498213

  8. Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients

    PubMed Central

    2014-01-01

    Background Alpha fetoprotein (AFP) is an oncofetal antigen over-expressed by many hepatocellular cancers (HCC). We previously demonstrated that HLA-A2-restricted epitopes derived from AFP are immunogenic in vitro and in vivo despite high circulating levels of this oncofetal antigen. In order to test a more broadly applicable, HLA-unrestricted, inexpensive, cell-free vaccine platform capable of activating tumor antigen-specific CD8+ and CD4+ T cells, we tested full length AFP in a plasmid DNA construct in combination with an AFP-expressing replication-deficient adenovirus (AdV) in a prime-boost vaccine strategy. Methods HCC patients who had an AFP+ tumor and previous treatment for HCC were screened and two patients received vaccination with three plasmid DNA injections followed by a single AdV injection, all delivered intramuscularly (i.m.). Results The vaccine was well tolerated and safe. Both patients showed immunologic evidence of immunization. The first patient had a weak AFP-specific T cell response, a strong AdV-specific cellular response and recurred with an AFP-expressing HCC at nine months. The second patient developed a strong AFP-specific CD8+ and CD4+ cellular response and an AdV neutralizing antibody response, and recurred at 18 months without an increase in serum AFP. Conclusions The AFP DNA prime-AdV boost vaccine was safe and immunogenic. Circulating anti-AdV neutralizing antibodies at baseline did not prohibit the development of AFP-specific cellular immunity. The patient who developed CD8+ and CD4+ AFP-specific T cell immunity had more favorable progression-free survival. The observations with these two patients support development of this vaccine strategy in a larger clinical trial. Trial registration ClinicalTrials.gov: NCT00093548 PMID:24708667

  9. 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.

  10. Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

    PubMed

    Li, D L; Huang, Y; Chang, L L; DU, Q; Chen, Y; Wang, T T; Luo, X M; Zhao, X M; Tong, D W

    2016-01-01

    Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2. PMID:27640437

  11. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

    PubMed Central

    Pascual, E.; Avia, M.; Rangel, G.; de Molina, A.; Alejo, A.; Sevilla, N.

    2016-01-01

    Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. PMID:26739058

  12. 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.

  13. 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

  14. 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

  15. [Rescue and Amplification of Recombinant Human Adenovirus Type 41 in 293 Cells].

    PubMed

    Zou, Xiaohui; Guo, Xiaojuan; Xiao, Rong; Wang, Min; Lu, Zhuozhuang; Hong, Tao

    2015-09-01

    Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 : 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells. PMID:26738289

  16. Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria.

    PubMed

    Morrison, Mark D; Reid, Dorothy; Onions, David; Spibey, Norman; Nicolson, Lesley

    2002-02-01

    E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs. A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1. PMID:11853396

  17. A New Type of Adenovirus Vector That Utilizes Homologous Recombination To Achieve Tumor-Specific Replication

    PubMed Central

    Bernt, Kathrin; Liang, Min; Ye, Xun; Ni, Shaoheng; Li, Zong-Yi; Ye, Sheng Long; Hu, Fang; Lieber, André

    2002-01-01

    We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application. PMID:12368342

  18. Priming T-cell responses with recombinant measles vaccine vector in a heterologous prime-boost setting in non-human primates.

    PubMed

    Bolton, Diane L; Santra, Sampa; Swett-Tapia, Cindy; Custers, Jerome; Song, Kaimei; Balachandran, Harikrishnan; Mach, Linh; Naim, Hussein; Kozlowski, Pamela A; Lifton, Michelle; Goudsmit, Jaap; Letvin, Norman; Roederer, Mario; Radošević, Katarina

    2012-09-01

    Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses.

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

    PubMed

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

    1998-01-01

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

  20. Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis.

    PubMed

    Maroof, Asher; Brown, Najmeeyah; Smith, Barbara; Hodgkinson, Michael R; Maxwell, Alice; Losch, Florian O; Fritz, Ulrike; Walden, Peter; Lacey, Charles N J; Smith, Deborah F; Aebischer, Toni; Kaye, Paul M

    2012-03-01

    Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.

  1. [Immortalization of rat corneal epithelial cells by SV40-adenovirus recombinant vector].

    PubMed

    Araki, K; Sasabe, T; Ohashi, Y; Yasuda, M; Handa, H; Tano, Y

    1994-04-01

    Using a SV40-adenovirus recombinant vector, we have successfully established a rat corneal epithelial cell line (RatCE) and studied its biological characteristics. RatCE continued to grow for more than 400 generations. It proliferated centrifugally in the early phase of the culture (1-3 days in culture) and had a cobblestone-like appearance in confluency. Desmosomes and microvilli were clearly seen under a transmission electron microscope. RatCE could be stored in liquid nitrogen and its biological characteristics were: doubling time, 18.3 hrs, colony forming ability, 36%, and growth ability in soft agar, 2%. When the insoluble extract from RatCE was electrophoresed, insoluble proteins were seen at 36 kD, 40 kD, 44 kD, 48 kD, 56 kD, and 64 kD. Anti-64 kD cytokeratin antibody strongly reacted with numerous filaments in the cytoplasm of RatCE. Hence, RatCE possessed 64 kD corneal specific keratin. A large amount of fibronectin was also assessed at focal contact by immunohistochemistry. Thus, RatCE retains several kinds of epithelial characteristics, is derived from one clone, and is immortalized. RatCE will be a useful tool in studies of the corneal epithelium. PMID:7513119

  2. Extended Protection against Phlebovirus Infection Conferred by Recombinant Adenovirus Expressing Consensus Interferon (DEF201)

    PubMed Central

    Ennis, Jane; Sefing, Eric J.; Wong, Min-Hui; Jung, Kie-Hoon; Turner, Jeffrey D.

    2012-01-01

    Punta Toro virus (PTV; Bunyaviridae, Phlebovirus) is related to Rift Valley fever virus (RVFV), a pathogenic agent which causes severe disease in humans and livestock primarily in the sub-Saharan region of Africa. The recent range expansion of RVFV and the potential for its intentional release into naïve populations pose a significant threat to public health and agriculture. Studies modeling disease in rodents and nonhuman primates have shown that PTV and RVFV are highly sensitive to the antiviral effects of alpha interferon (IFN-α), an important component of the innate antiviral host response. While recombinant IFN-α has high therapeutic value, its utility for the treatment of neglected tropical diseases is hindered by its short in vivo half-life and costly production of longer-lasting pegylated IFNs. Here, we demonstrate extended preexposure protection against lethal PTV challenge following a single intranasal administration of DEF201, which is a replication-deficient human adenovirus type 5 vector engineered to constitutively express consensus IFN-α (cIFN-α) from transduced host cells. DEF201 was also efficacious when administered within 24 h as a postexposure countermeasure. Serum concentrations of cIFN-α could be detected as early as 8 h following treatment and persisted for more than 1 week. The prolonged antiphlebovirus prophylactic effect, low production costs, and ease of administration make DEF201 a promising agent for intervention during natural disease outbreaks and for countering possible bioterrorist acts. PMID:22615273

  3. Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice.

    PubMed

    Maunder, Helen E; Taylor, Geraldine; Leppard, Keith N; Easton, Andrew J

    2015-11-27

    Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8(+) T cell response and, to a lesser extent, a CD4(+) T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.

  4. 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

  5. Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

    SciTech Connect

    Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian

    2007-10-05

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10{sup 8} PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization.

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

    PubMed

    He, Hong; Zhang, Jieqiong; Fan, Yan; Sun, Xiaoshuang; Zhu, Yuhao

    2016-01-01

    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

  7. 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

  8. Effects of recombinant human adenovirus-p53 on the regression of hepatic fibrosis

    PubMed Central

    Liu, Yehong; Yang, Puye; Chen, Na; Lin, Shumei; Liu, Min

    2016-01-01

    Hepatic fibrosis is a scarring process that may progress to hepatic cirrhosis and even hepatic carcinoma if left untreated. Hepatic stellate cells (HSCs) play essential roles in the development of hepatic fibrosis. The tumor suppressor protein p53 is a transcription factor that is involved in cell proliferation, cell cycle regulation, apoptosis and DNA repair. Recombinant human adenovirus-p53 (Ad-p53) has been demonstrated to act as a promising antitumor gene therapy in various types of cancer. However, there is limited infomration regarding the therapeutic effect of Ad-p53 on the regression of hepatic fibrosis. In order to examine the underlying molecular mechanism responsible for the effects of Ad-p53 on HSCs, a rat model of hepatic fibrosis was established and HSC-T6 cells were cultured under different conditions. The expression of p53, transforming growth factor (TGF-β1) and α-smooth muscle actin (α-SMA), which is a marker of activated HSCs, was detected by immunohistochemical assays and RT-qPCR. In vitro, five different concentrations (1×106, 5×106, 1×107, 2×107 and 5×107 PFU/ml) of Ad-p53 were selected for use in the MTT assay to analyze the proliferation of HSCs at 0, 24, 48 and 72 h. Flow cytometric analysis was applied to determine the effect of three different concentrations of Ad-p53 (5×106, 1×107 and 2×107 PFU/ml) on the cell cycle and the apoptosis of HSC-T6 cells at 24 and 48 h. The results of immunohistochemical studies and RT-qPCR showed that Ad-p53 upregulated the expression of p53, and downregulated the expression of TGF-β1 and α-SMA. The MTT assay revealed that when treated with various doses of Ad-p53, the proliferation of HSCs was inhibited within a certain range of concentrations and time periods. Analysis of flow cytometric data showed that Ad-p53 arrested the cell cycle in G1 phase and significantly induced apoptosis. Taken together, these findings suggest that Ad-p53 promotes apoptosis and inhibits the proliferation of HSCs in

  9. Application of a Fas Ligand Encoding a Recombinant Adenovirus Vector for Prolongation of Transgene Expression

    PubMed Central

    Zhang, Huang-Ge; Bilbao, Guadalupe; Zhou, Tong; Contreras, Juan Luis; Gómez-Navarro, Jesús; Feng, Meizhen; Saito, Izumu; Mountz, John D.; Curiel, David T.

    1998-01-01

    An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity. PMID:9499110

  10. Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

    PubMed Central

    Kosinska, Anna D.; Zhang, Ejuan; Johrden, Lena; Liu, Jia; Seiz, Pia L.; Zhang, Xiaoyong; Ma, Zhiyong; Kemper, Thekla; Fiedler, Melanie; Glebe, Dieter; Wildner, Oliver; Dittmer, Ulf; Lu, Mengji; Roggendorf, Michael

    2013-01-01

    A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients. PMID:23785279

  11. Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep.

    PubMed

    Rojas, José M; Moreno, Héctor; Valcárcel, Félix; Peña, Lourdes; Sevilla, Noemí; Martín, Verónica

    2014-01-01

    Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

  12. 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

  13. 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.

  14. 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

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

    PubMed

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine. PMID:19178894

  16. Substrate oscillations boost recombinant protein release from Escherichia coli.

    PubMed

    Jazini, Mohammadhadi; Herwig, Christoph

    2014-05-01

    Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.

  17. 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

  18. 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.

  19. Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75

    PubMed Central

    Kumar, Ramesh; Sreenivasa, B. P.; Tamilselvan, R. P.

    2015-01-01

    Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV) capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm). Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5). Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01). Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by

  20. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  1. [Construction of a Recombinant Replication-defective Human Adenovirus Type 5 Expressing G Protein of Irkut Virus and the Immune Test in Mouse].

    PubMed

    Wang, Yuying; Chen, Qi; Liu, Ye; Hu, Rongliang; Zhang, Lecui

    2015-11-01

    To develop a safe and effective new generation vaccine for IRKV-THChina12 prevention, we constructed a non-replicative recombinant human adenovirus carrying the IRKV-THChina12 G gene, named as rAd5-IRKV-G. The IRKV-THChina12 G protein expressed by the recombinant human adenovirus in 293AD cells was detected by western blot and indirect immunofluorescence test. To evaluate the immunogenicity of the recombinant, mice were immunized with rAd5-IRKV-G by intramuscular (i. m.) or intraperitoneal (i. p.) route and with non-exogenous gene expressing wild type adenovirus wt-rAd5 as a control. Results showed that the rAd5-IRKV-G could induce continuous and statistically significant (P ≤ 0.05) anti-IRKV neutralizing antibody (NA) production in immunized mice by i. m. or i. p. route. In particular, no significant difference (P > 0.05) of the NA titers between the two administration routes were observed, that provides an alternative choice for animal immunization method in the future application. PMID:26951008

  2. DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1.

    PubMed

    Li, Junping; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Feng, Yufei; Lv, Shuang; Zhang, Qin; Wang, Haixiu; Wu, Donglai

    2015-10-01

    Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

  3. Gene therapy for human colorectal cancer cell lines with recombinant adenovirus 5 based on loss of the insulin-like growth factor 2 imprinting.

    PubMed

    Sun, Huiling; Pan, Yuqin; He, Bangshun; Deng, Qiwen; Li, Rui; Xu, Yeqiong; Chen, Jie; Gao, Tianyi; Ying, Houqun; Wang, Feng; Liu, Xian; Wang, Shukui

    2015-04-01

    The recombinant oncolytic adenovirus is a novel anticancer agent to replicate selectively in colon cancer cell lines. Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon. We utilized the IGF2 LOI in gene therapy for the malignant tumor cell lines. We investigated the tumoricidal effects of IGF2 LOI on four cell lines by oncolytic adenovirus, and constructed novel adenovirus vectors Ad312-E1A and Ad312-EGFP. The expression of E1A was monitored by real-time PCR and western blot analysis. The viability and apoptosis of colorectal cells infected with Ad312-E1A were tested by CCK-8 and flow cytometry. In addition, we established a colorectal cancer model in nude mice. The results showed that HCT-8 and HT-29 with IGF2 LOI were infected with Ad312-EGFP and then produced the EGFP. Nevertheless, SW480 and GES-1, which were IGF2 MOI, did not produce the EGFP. The Ad312-E1A obviously reduced the cell viability and induced apoptosis in HCT-8 and HT-29 in vitro, and successfully suppressed tumor growth in HT-29 xenografts in nude mice. In summary, the conditionally replicative adenovirus with loss of IGF2 imprinting system has a positive effect on gene therapy.

  4. 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

  5. Immune responses of recombinant adenovirus co-expressing VP1 of foot-and-mouth disease virus and porcine interferon alpha in mice and guinea pigs.

    PubMed

    Du, Yijun; Dai, Jianjun; Li, Yufeng; Li, Congzhi; Qi, Jing; Duan, Shuyi; Jiang, Ping

    2008-08-15

    Foot-and-mouth disease (FMD) is a highly contagious and economically devastating vesicular disease of cloven-hoofed animals. In this study, we constructed and characterized the immune responses and vaccine efficacy conferred by the recombinant adenovirus co-expressing VP1 of FMDV and porcine interferon alpha as fusion protein (rAd-pIFNalpha-VP1). Six groups of female BALB/c mice each with 18 were inoculated subcutaneously twice 2-week intervals with the recombinant adenoviruses. The results showed that the levels of humoral and cell-mediated immune responses in the group inoculated with rAd-pIFNalpha-VP1 were significantly higher than those in the group inoculated with rAd-VP1+rAd-pIFNalpha (P<0.05). Then four groups of guinea pigs each with six were inoculated two times at 2-week intervals intramuscularly with rAd-pIFNalpha-VP1, commercial inactivated FMD vaccine, wild-type adenovirus (wtAd) or PBS, and the protective efficacy of rAd-pIFNalpha-VP1 was determined. The results indicated that all the guinea pigs vaccinated with rAd-pIFNalpha-VP1 as well as inactivated FMD vaccine were protected from FMDV challenge, even though the levels of neutralizing antibodies (1:32-1:40) of the animals vaccinated with rAd-pIFNalpha-VP1 was lower than that in the group inoculated with inactivated FMD vaccine (1:64-1:128). It demonstrated that the newly recombinant adenovirus rAd-pIFNalpha-VP1 might further be an attractive candidate vaccine for preventing FMDV infection in swine. PMID:18511133

  6. 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

  7. 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.

  8. Immunoglobulin genes and the acquisition of HIV infection in a randomized trial of recombinant adenovirus HIV vaccine.

    PubMed

    Pandey, Janardan P; Namboodiri, Aryan M; Bu, Shizhong; De Dieu Tapsoba, Jean; Sato, Alicia; Dai, James Y

    2013-06-20

    Our knowledge of the host genetic factors that contribute to the acquisition of HIV infection is limited. To identify the host genetic correlates of HIV1 acquisition, we genotyped 777 participants of a randomized trial of recombinant adenovirus HIV1 vaccine for Fcγ receptor IIa (FcγRIIa), FcγRIIIa, and several GM and KM alleles-genetic markers of immunoglobulin γ and κ chains, respectively. None of the genotypes by itself was significantly associated with the acquisition of HIV1 infection. However, particular combinations of GM and KM as well as those of GM and FcγRIIIa loci were significantly associated with the acquisition of HIV1 infection epistatically: KM1/3-GM3/17 (interaction p=0.0246; FDR=0.2952), KM1/3-GM5/21 (interaction p=0.0016; FDR=0.0960), and GM23+/-FcγRIIIa (interaction p=0.0060; FDR=0.1200). These results suggest the involvement of GM, KM, and FcγRIIIa loci in the acquisition of HIV infection. Additional studies are warranted.

  9. Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

    PubMed Central

    Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J.; Farness, Peggy; Avanzini, Jenny B.; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J.; Mayall, Tim

    2012-01-01

    Background Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. Methodology/Principal Findings The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Conclusions/Significance Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine. PMID:22363572

  10. A single immunization with a recombinant canine adenovirus type 2 expressing the seoul virus Gn glycoprotein confers protective immunity against seoul virus in mice.

    PubMed

    Yuan, Zi-Guo; Li, Xiu-Ming; Mahmmod, Yasser Saad; Wang, Xiao-Hu; Xu, Hui-Juan; Zhang, Xiu-Xiang

    2009-08-20

    Seoul virus (SEOV), a member of hantavirus genus, is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) and afflicts tens of thousands of people annually. In this paper, we evaluate the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gn protein of SEOV (rCAV-2-Gn) in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against a lethal challenge with the highly virulent SEOV strain CC-2. Protective level neutralizing antibodies were maintained for at least 20 weeks. The efficacy of the recombinant was similar to that induced by a currently available inactivated HFRS vaccine. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

  11. 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...

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. In vivo correction with recombinant adenovirus of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in strain III mice.

    PubMed

    Kubo, S; Kiwaki, K; Awata, H; Katoh, H; Kanegae, Y; Saito, I; Yamamoto, T; Miyazaki, J; Matsuda, I; Endo, F

    1997-01-01

    Tyrosinemia type 3, caused by a genetic deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (HPD) in tyrosine catabolism, is characterized by convulsion, ataxia, and mental retardation. The III mouse is a model of tyrosinemia type 3. HPD activity and protein are defective in the liver and its blood tyrosine levels are elevated, the range being between 1,100 and 1,656 microM. We constructed a recombinant adenoviral vector bearing the human HPD cDNA (AdexCAGhHPD), which is expressed under the control of a potent CAG promoter. III mice were injected with 1.0 x 10(8) to 1.0 x 10(9) pfu of AdexCAGhHPD through the tail vein. When 3.0 x 10(8) - 1.0 x 10(9) pfu were injected, blood tyrosine levels decreased within 3 hr, reached a normal range (under 300 microM), and remained at a low level for 2-6 weeks. Hepatic HPD activities also increased as early as 3 hr after the injection of 5.0 x 10(8) pfu, reached the levels comparable to the control mice in 3-7 days, and then decreased, and correlated well to blood tyrosine. Hepatic HPD expression was confirmed by Northern blot and immunoblot analyses. Histology revealed no difference (gross or microscopic) between the liver injected with AdexCAGhHPD and the control. No significant changes in blood tyrosine levels were noted after the second injection of 5.0 x 10(8) pfu of AdexCAGhHPD. Thus, the intravenous administration of the adenoviral vector bearing a foreign gene seems suitable for transient, early gene transfer into the liver.

  18. Immune responses of two recombinant adenoviruses expressing VP1 antigens of FMDV fused with porcine granulocyte macrophage colony-stimulating factor.

    PubMed

    Du, Yijun; Jiang, Ping; Li, Yufeng; He, Hairong; Jiang, Wenming; Wang, Xinglong; Hong, Weibin

    2007-11-28

    Foot-and-mouth disease (FMD) is a highly contagious and economically devastating vesicular disease of cloven-hoofed animals. In the present report, we constructed and characterized the immune responses conferred by two recombinant adenoviruses expressing VP1 epitopes (three amino acid residues 21-60, 141-160 and 200-213 in VP1, designated VPe) or VP1 protein of FMDV fused with porcine granulocyte macrophage colony-stimulating factor (named rAd-GMCSF-VPe and rAd-GMCSF-VP1). Seven groups of female BALB/c mice each containing 18 mice were inoculated subcutaneously twice at 2-week intervals with the recombinant adenoviruses. Then the protective efficacy of the two adenoviruses was detected in guinea pigs and swine. The results showed that the highest levels of FMDV-specific T cell proliferation, IFN-gamma and IL-4 could be induced by rAd-GMCSF-VPe expressing fusion GMCSF-VPe, and the highest level of FMDV-specific humoral immune responses could be induced by rAd-GMCSF-VP1 expressing fusion GMCSF-VP1 in mice. All guinea pigs and swine co-administrated with rAd-GMCSF-VPe and rAd-GMCSF-VP1 were protected from viral challenge, even though the neutralizing antibody titers were significantly lower than those in the group inoculated with inactivated FMD vaccine. It demonstrated that co-administration of rAd-GMCSF-VPe and rAd-GMCSF-VP1 might be attractive candidate vaccines for preventing FMDV infection. PMID:17980939

  19. Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

    PubMed

    O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

    2014-03-01

    The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

  20. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

    PubMed

    Cao, Shinuo; Mousa, Ahmed Abdelmoniem; Aboge, Gabriel Oluga; Kamyingkird, Ketsarin; Zhou, Mo; Moumouni, Paul Franck Adjou; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Fukumoto, Shinya; Xuan, Xuenan

    2013-12-01

    A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs. PMID:24338330

  1. 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 ...

  2. 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

  3. Treatment of Parkinson disease with C17.2 neural stem cells overexpressing NURR1 with a recombined republic-deficit adenovirus containing the NURR1 gene.

    PubMed

    Li, Qing-Jun; Tang, Ya-Mei; Liu, Jun; Zhou, Dao-You; Li, Xiang-Pen; Xiao, Song-Hua; Jian, Dong-Xing; Xing, Yi-Gang

    2007-12-01

    To study the potential benefit of the NURR1 gene in Parkinson's disease (PD), we constructed a recombinant republic-deficit adenovirus containing the NURR1 gene (Ad-NURR1) and expressed it in transplanted neural stem cells (NSC). Ad-NURR1 was constructed, and NURR1 mRNA and protein expression were identified by in situ hybridization and western blot analysis, respectively. The identified NURR1 protein could directly or indirectly induce NSC differentiation into neurons. To identify a potential therapeutic use for the transfected NSCs, cells were transplanted into 6-hydroxydopamine lesioned rats. Histopathological and behavioral alterations were evaluated via immunohistochemistry and the ration test, respectively, in rats transplanted with NSCs with or without the Ad-NURR1 adenovirus. The Ad-NURR1 construct effectively expressed the NURR1 protein, which could directly or indirectly induce NSC differentiation into neurons. Both histopathological and behavioral alterations were seen in rats treated with NSCs with or without the Ad-NURR1 construct, although in the case of the latter, the benefits were more robust. These results suggest a potential therapeutic benefit for Ad-NURR1-expressing cells in the treatment of PD. The Ad-NURR1 modification induced NSC differentiation and therefore represents a potential therapy for PD.

  4. Recombination of the Epsilon Determinant and Corneal Tropism: Human Adenovirus Species D Types 15, 29, 56, and 69

    PubMed Central

    Singh, Gurdeep; Zhou, Xiaohong; Lee, Jeong Yoon; Yousuf, Mohammad A.; Ramke, Mirja; Ismail, Mohamed A.; Lee, Ji Sun; Robinson, Christopher M.; Seto, Donald; Dyer, David W.; Jones, Morris S.; Rajaiya, Jaya; Chodosh, James

    2015-01-01

    Viruses within human adenovirus species D (HAdV-D) infect epithelia at essentially every mucosal site. Hypervariable loops 1 and 2 of the hexon capsid protein contain epitopes that together form the epsilon determinant for serum neutralization. We report our analyses comparing HAdV-D15, 29, 56, and the recently identified type 69, each with highly similar hexons and the same serum neutralization profile, but otherwise disparate genomes. Of these, only HAdV-D type 56 is associated with epidemic keratoconjunctivitis (EKC), a severe infection of ocular surface epithelium and underlying corneal stroma. In the mouse adenovirus keratitis model, all four viruses induced inflammation. However, HAdV-D56 entry into human corneal epithelial cells and fibroblasts in vitro dramatically exceeded that of the other three viruses. We conclude that the hexon epsilon determinant is not a prime contributor to corneal tropism. PMID:26343864

  5. 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.

  6. 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.

  7. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques

    PubMed Central

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 108 PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4. PMID:26718849

  8. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

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

  9. 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

    2014-02-26

    Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that 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.

  10. 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 Central

    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

  11. Experimental oral immunization of ferret badgers (Melogale moschata) with a recombinant canine adenovirus vaccine CAV-2-E3Δ-RGP and an attenuated rabies virus SRV9.

    PubMed

    Zhao, Jinghui; Liu, Ye; Zhang, Shoufeng; Fang, Lijun; Zhang, Fei; Hu, Rongliang

    2014-04-01

    Ferret badgers (Melogale moschata) are a major reservoir of rabies virus in southeastern China. Oral immunization has been shown to be a practical method for wildlife rabies management in Europe and North America. Two groups of 20 ferret badgers were given a single oral dose of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3Δ-RGP, or an experimental attenuated rabies virus vaccine, SRV9. At 21 days, all ferret badgers had seroconverted, with serum virus-neutralizing antibodies ranging from 0.1 to 4.5 IU/mL. Titers were >0.50 IU/mL (an acceptable level) in 17/20 and 16/20 animals receiving CAV-2-E3Δ-RGP or SRV9, respectively. The serologic results indicate that the recombinant CAV-2-E3Δ-RGP is at least as effective as the attenuated rabies virus vaccine. Both may be considered for additional research as oral rabies vaccine candidates for ferret badgers.

  12. Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

    PubMed

    Aravind, S; Kamble, Nitin Machindra; Gaikwad, Satish S; Shukla, Sanjeev Kumar; Saravanan, R; Dey, Sohini; Mohan, C Madhan

    2015-11-01

    Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

  13. Potent Functional Antibody Responses Elicited by HIV-I DNA Priming and Boosting with Heterologous HIV-1 Recombinant MVA in Healthy Tanzanian Adults

    PubMed Central

    Joachim, Agricola; Nilsson, Charlotta; Aboud, Said; Bakari, Muhammad; Lyamuya, Eligius F.; Robb, Merlin L.; Marovich, Mary A.; Earl, Patricia; Moss, Bernard; Ochsenbauer, Christina; Wahren, Britta; Mhalu, Fred; Sandström, Eric; Biberfeld, Gunnel; Ferrari, Guido; Polonis, Victoria R.

    2015-01-01

    Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA) virus boosting (HIVIS03). The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC) assay using luciferase reporter-infectious molecular clones (LucR-IMC) was employed. The serum neutralizing activity was significantly (but not completely) reduced upon depletion of natural killer (NK) cells from PBMC (p=0.006), indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development. Trial Registration Controlled-Trials ISRCTN90053831 The Pan African Clinical Trials Registry ATMR2009040001075080 (currently PACTR2009040001075080) PMID:25874723

  14. Bypassing tumor-associated immune suppression with recombinant adenovirus constructs expressing membrane bound or secreted GITR-L.

    PubMed

    Calmels, Bastien; Paul, Stéphane; Futin, Nicolas; Ledoux, Catherine; Stoeckel, Fabienne; Acres, Bruce

    2005-02-01

    Recent evidence has resurrected the concept of specialized populations of T lymphocytes that are able to suppress an antigen-specific immune response. T-regulatory cells (T-reg) have been characterized as CD4+ CD25+ T cells. Previous reports describing differential gene expression analysis have shown that the glucocorticoid-induced tumor necrosis family receptor family-related gene (GITR) is upregulated in these cells. Furthermore, antibodies specific for GITR have been shown to inhibit the T-suppressor function of CD4+ CD25+ T-reg. The ligands for both mouse and human GITR have been cloned recently. We have inserted the sequences for natural, membrane-bound GITR-ligand (GITR-L) and a truncated secreted form of GITR-L (GITR-Lsol) into the adenovirus-5 genome. Coculture experiments show that cells infected with Ad-GITR-L and supernatants from cells infected with Ad-GITR-Lsol can increase the proliferation of both CD4+ CD25- and CD8+ T cells in response to anti-CD3 stimulation, in the presence, as well as in the absence, of CD4+ CD25+ T cells. The virus constructs were injected into growing B16 melanoma tumors. Ad-GITR-L was shown to attract infiltration with both CD4+ and CD8+ T cells. Both constructs were shown to inhibit tumor growth. PMID:15472713

  15. Recombinant adenovirus snake venom cystatin inhibits the growth, invasion, and metastasis of B16F10 cells in vitro and in vivo.

    PubMed

    Xie, Qun; Tang, Nanhong; Lin, Yangyuan; Wang, Xiaoqian; Lin, Xu; Lin, Jianyin

    2013-12-01

    Previous studies have shown that transfection of the snake venom cystatin (sv-cystatin) gene can inhibit the invasion and metastasis of tumor cells. The aim of this study was to investigate the pharmaceutical applications of sv-cystatin in melanoma gene therapy. We constructed a recombinant adenovirus carrying sv-cystatin (Ad/sv-cystatin) and a control virus (Ad/null). Matrigel assays were used to assess melanoma cell migration and invasiveness in vitro. The antimelanoma effects of Ad/sv-cystatin were assessed in a syngeneic mouse model with an experimental lung colonization assay. Ad/sv-cystatin significantly inhibited the invasion and growth of B16F10 cells in vitro compared with control and Ad/null. Ad/sv-cystatin significantly inhibited experimental lung colonization in C57BL/6 mice as compared with that in control (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 51 and 46%, respectively. Ad/sv-cystatin slowed the increase in lung weight in C57BL/6 mice as compared with that in control mice (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 40 and 35%, respectively. Our results indicate that Ad/sv-cystatin suppresses mouse melanoma invasion, metastasis, and growth in vitro and in vivo. Our findings provide support for the further examination of the pharmaceutical applications of Ad/sv-cystatin.

  16. Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens.

    PubMed

    R, Rajasekhar; Roy, Parimal

    2014-10-01

    A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Fowl adenovirus-4 (FAdV) causing Hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737bp was cloned into pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested by Western Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4 serology. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen was also used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometric mean (GM) antibody titres as measured by ELISA ranged from 5.006±0.11log10 to 4.526±0.04log10 and by Dot ELISA titre were from 2.240±0.08log10 to 0.180±0.04log10 during 5th-8th weeks of age, results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks, found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positive at 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have considerably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages of recombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed.

  17. 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

  18. A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

    PubMed

    Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Zhao, Chunxia; Makarova, Natalia; Dmitriev, Igor; Curiel, David T; Blackwell, Jerry; Moreno, Alberto

    2016-10-01

    An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development. PMID:27574299

  19. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    PubMed

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

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  20. 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

  1. Effect of UV light on the inactivation of recombinant human adenovirus and murine norovirus seeded in seawater in shellfish depuration tanks.

    PubMed

    Garcia, Lucas A T; Nascimento, Mariana A; Barardi, Célia R M

    2015-03-01

    Shellfish depuration is a process that aims to eliminate pathogens from mollusk tissues. Seawater disinfection during the depuration process is important and ultraviolet (UV) light treatment is the most used method worldwide. Viral models are usually employed as surrogates of fastidious viruses in viability studies. The aim of this study was to employ methods based on green fluorescent protein (GFP) fluorescence and plaque forming units to detect, respectively, recombinant adenovirus (rAdV-GFP) and murine norovirus (MNV) artificially seeded in environmental matrices. These assays were applied to assess the inactivation of rAdV-GFP and MNV in seawater in recirculation shellfish depuration tanks with and without UV light treatment. Kinetics of rAdV GFP-expression was previously measured by UV-spectrophotometer. Flow cytometry (FC), fluorescence microscopy (FM), and plaque assay were used to determine virus titer and detection limits. The influence of the environmental matrix on the performance of the methods was prior determined using either drinking water or filtered seawater seeded with rAdV-GFP. Disinfection of seeded seawater was evaluated with and without UV treatment. The time of 24-h post-infection was established as ideal for fluorescence detection on rAdV-GFP infected cells. FC showed lower sensitivity, when compared to FM, which was similar to plaque assay. Seawater disinfection on depuration tanks was promising and rAdV-GFP declined 99.99 % after 24 and 48 h with and without UV treatment, respectively. MNV was completely inactivated after 24 h in both treatments. As conclusion, the depuration tanks were effective to inactivate rAdV-GFP and MNV and the UV disinfection treatment accelerated the process.

  2. 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

  3. Killing effect of Ad5/F35-APE1 siRNA recombinant adenovirus in combination with hematoporphrphyrin derivative-mediated photodynamic therapy on human nonsmall cell lung cancer.

    PubMed

    Xia, Lei; Guan, Wei; Wang, Dong; Zhang, Yun-Song; Zeng, Lin-Li; Li, Zeng-Peng; Wang, Ge; Yang, Zhen-Zhou

    2013-01-01

    The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

  4. 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

  5. Mesenchymal Stromal Cells Engineered to Produce IGF-I by Recombinant Adenovirus Ameliorate Liver Fibrosis in Mice

    PubMed Central

    Fiore, Esteban J.; Bayo, Juan M.; Garcia, Mariana G.; Malvicini, Mariana; Lloyd, Rodrigo; Piccioni, Flavia; Rizzo, Manglio; Peixoto, Estanislao; Sola, M. Beatriz; Atorrasagasti, Catalina; Alaniz, Laura; Camilletti, María A.; Enguita, Mónica; Prieto, Jesús; Aquino, Jorge B.

    2015-01-01

    Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis. PMID:25315017

  6. Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.

    PubMed

    Fiore, Esteban J; Bayo, Juan M; Garcia, Mariana G; Malvicini, Mariana; Lloyd, Rodrigo; Piccioni, Flavia; Rizzo, Manglio; Peixoto, Estanislao; Sola, M Beatriz; Atorrasagasti, Catalina; Alaniz, Laura; Camilletti, María A; Enguita, Mónica; Prieto, Jesús; Aquino, Jorge B; Mazzolini, Guillermo

    2015-03-15

    Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.

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

    PubMed

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

    2013-04-01

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

  8. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection.

  9. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection. PMID:27177947

  10. 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

  11. A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers.

    PubMed

    Radaelli, Antonia; De Giuli Morghen, Carlo; Zanotto, Carlo; Pacchioni, Sole; Bissa, Massimiliano; Franconi, Rosella; Massa, Silvia; Paolini, Francesca; Muller, Antonio; Venuti, Aldo

    2012-12-01

    Development of effective therapeutic vaccines against human papilloma virus (HPV) infections remains a priority, considering the high number of new cases of cervical cancer each year by high-risk HPVs, in particular by HPV-16. Vaccines expressing the E7 oncoprotein, which is detectable in all HPV-positive pre-cancerous and cancer cells, might clear already established tumors and support the treatment of HPV-related lesions. In this study, DNA or fowlpox virus recombinants expressing the harmless variant E7GGG of the HPV-16 E7 oncoprotein (DNA(E7GGG) and FP(E7GGG)) were generated. Two immunization regimens were tested in a pre-clinical mouse model by homologous (FP/FP) or heterologous (DNA/FP) prime-boost protocols to evaluate the immune response and therapeutic efficacy of the proposed HPV-16 vaccine. Low levels of anti-E7-specific antibodies were elicited after immunization, and in vivo experiments resulted in a higher number of tumor-free mice after the heterologous immunization. These results establish a preliminary indication for therapy of HPV-related tumors by the combined use of DNA and avipox recombinants, which might represent safer immunogens than vaccinia-based vaccines.

  12. Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

    PubMed Central

    Saljoughian, Noushin; Taheri, Tahereh; Zahedifard, Farnaz; Taslimi, Yasaman; Doustdari, Fatemeh; Bolhassani, Azam; Doroud, Delaram; Azizi, Hiva; Heidari, Kazem; Vasei, Mohammad; Namvar Asl, Nabiollah; Papadopoulou, Barbara; Rafati, Sima

    2013-01-01

    Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. PMID:23638195

  13. Protective MCMV immunity by vaccination of the salivary gland via Wharton's duct: replication-deficient recombinant adenovirus expressing individual MCMV genes elicits protection similar to that of MCMV.

    PubMed

    Liu, Guangliang; Zhang, Fangfang; Wang, Ruixue; London, Lucille; London, Steven D

    2014-04-01

    Salivary glands, a major component of the mucosal immune system, confer antigen-specific immunity to mucosally acquired pathogens. We investigated whether a physiological route of inoculation and a subunit vaccine approach elicited MCMV-specific and protective immunity. Mice were inoculated by retrograde perfusion of the submandibular salivary glands via Wharton's duct with tcMCMV or MCMV proteins focused to the salivary gland via replication-deficient adenovirus expressing individual MCMV genes (gB, gH, IE1; controls: saline and replication deficient adenovirus without MCMV inserts). Mice were evaluated for MCMV-specific antibodies, T-cell responses, germinal center formation, and protection against a lethal MCMV challenge. Retrograde perfusion with tcMCMV or adenovirus expressed MCMV proteins induced a 2- to 6-fold increase in systemic and mucosal MCMV-specific antibodies, a 3- to 6-fold increase in GC marker expression, and protection against a lethal systemic challenge, as evidenced by up to 80% increased survival, decreased splenic pathology, and decreased viral titers from 10(6) pfu to undetectable levels. Thus, a focused salivary gland immunization via a physiological route with a protein antigen induced systemic and mucosal protective immune responses. Therefore, salivary gland immunization can serve as an alternative mucosal route for administering vaccines, which is directly applicable for use in humans.

  14. 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

  15. Replication-Competent Adenovirus Formation in 293 Cells: the Recombination-Based Rate Is Influenced by Structure and Location of the Transgene Cassette and Not Increased by Overproduction of HsRad51, Rad51-Interacting, or E2F Family Proteins

    PubMed Central

    Duigou, Gregory J.; Young, C. S. H.

    2005-01-01

    Propagation of E1 region replacement adenovirus vectors in 293 cells results in the rare appearance of replication-competent adenovirus (RCA). The RCA genome contains E1 DNA acquired from the 293 cellular genome. The Luria-Delbrück fluctuation test was adapted to measure RCA formation rates. To test if structure affected rate, we measured rates during the production of adenovirus vectors with genomes containing three different expression cassette arrangements. The vectors had different extents of sequence identity with integrated Ad5 DNA of 293 cells and had different distributions of identity flanking the expression cassettes. Empty cassette vector RCA rates ranged from 2.5 × 10−8 to 5.6 × 10−10. The extent of sequence identity was not an accurate RCA rate predictor. The vector with the highest RCA rate also had the least overall sequence identity. To define factors controlling RCA generation, adenovirus vectors expressing E2F family proteins, known to modulate recombination gene expression, and overexpressing the human Rad51 recombination protein were analyzed. Compared to their corresponding empty vectors, RCA rates were not increased but were slightly decreased. Initial results suggested expression cassette orientation and/or transcription direction as potential RCA rate modifiers. Testing adenovirus vectors with identical transgene cassettes oriented in opposite directions suggested that transcription direction was not the basis of these rate differences. Thus, the overall structure and location of the transgene cassette had the largest effect on RCA rate. The RCA fluctuation test should be useful for investigators who require accurate measurements of targeted recombination and the probability of RCA formation during stock production. PMID:15827158

  16. 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

  17. 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

  18. 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

  19. 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

  20. Induction of HIV-1–Specific Mucosal Immune Responses Following Intramuscular Recombinant Adenovirus Serotype 26 HIV-1 Vaccination of Humans

    PubMed Central

    Baden, Lindsey R.; Liu, Jinyan; Li, Hualin; Johnson, Jennifer A.; Walsh, Stephen R.; Kleinjan, Jane A.; Engelson, Brian A.; Peter, Lauren; Abbink, Peter; Milner, Danny A.; Golden, Kevin L.; Viani, Kyle L.; Stachler, Matthew D.; Chen, Benjamin J.; Pau, Maria G.; Weijtens, Mo; Carey, Brittany R.; Miller, Caroline A.; Swann, Edith M.; Wolff, Mark; Loblein, Hayley; Seaman, Michael S.; Dolin, Raphael; Barouch, Dan H.

    2015-01-01

    Background Defining mucosal immune responses and inflammation to candidate human immunodeficiency virus type 1 (HIV-1) vaccines represents a current research priority for the HIV-1 vaccine field. In particular, it is unclear whether intramuscular immunization can elicit immune responses at mucosal surfaces in humans. Methods In this double-blind, randomized, placebo-controlled clinical trial, we evaluated systemic and mucosal immune responses to a candidate adenovirus serotype 26 (Ad26) vectored HIV-1 envelop (Env) vaccine in baseline Ad26-seronegative and Ad26-seropositive healthy volunteers. Systematic mucosal sampling with rectal Weck-Cel sponges and rectal biopsies were performed. Results Intramuscular immunization elicited both systemic and mucosal Env-specific humoral and cellular immune responses in the majority of subjects. Individuals with preexisting Ad26-specific neutralizing antibodies had vaccine-elicited immune responses comparable to those of subjects who were Ad26 seronegative. We also observed no increase in activated total or vector-specific mucosal CD4+ T lymphocytes following vaccination by either histopathology or flow cytometry. Conclusions These data demonstrate that a single intramuscular administration of this Ad26-vectored HIV-1 Env vaccine elicited both systemic and mucosal immune responses in humans. Induction of antigen-specific humoral and cellular mucosal immunity was not accompanied by a detectable increase in mucosal inflammation. Clinical Trials Registration NCT01103687. PMID:25165165

  1. 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

  2. Evaluation of different heterologous prime-boost immunization strategies against Babesia bovis using viral vectored and protein-adjuvant vaccines based on a chimeric multi-antigen.

    PubMed

    Jaramillo Ortiz, José Manuel; Molinari, María Paula; Gravisaco, María José; Paoletta, Martina Soledad; Montenegro, Valeria Noely; Wilkowsky, Silvina Elizabeth

    2016-07-19

    Protection against the intraerythrocytic bovine parasite Babesia bovis requires both humoral and cellular immune responses. Therefore, tailored combinations of immunogens targeted at both arms of the immune system are strategies of choice to pursue sterilizing immunity. In this study, different heterologous prime-boost vaccination schemes were evaluated in mice to compare the immunogenicity induced by a recombinant adenovirus, a modified vaccinia Ankara vector or a subunit vaccine all expressing a chimeric multi-antigen. This multi-antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: Merozoite Surface Antigen - 2c (MSA-2c), Rhoptry Associated Protein - 1 (RAP-1) and Heat Shock Protein 20 (HSP20). Both priming with the adenovirus or recombinant multi-antigen and boosting with the modified vaccinia Ankara vector achieved a high degree of activation of TNFα and IFNγ-secreting CD4(+) and CD8(+) specific T cells 60days after the first immunization. High titers of specific IgG antibodies were also detected at the same time point and lasted up to day 120 of the first immunization. Only the adenovirus - MVA combination triggered a marked isotype skew for the IgG2a antibody subclass meanwhile for the other immune traits analyzed here, both vaccination schemes showed similar performances. The immunological characterization in the murine model of these rationally designed immunogens led us to propose that adenoviruses as well as the bacterially expressed multi-antigen are highly reliable primer candidates to be considered in future experiments in cattle to test protection against bovine babesiosis. PMID:27269058

  3. Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus

    PubMed Central

    Robinson, Christopher M.; Zhou, Xiaohong; Rajaiya, Jaya; Yousuf, Mohammad A.; Singh, Gurdeep; DeSerres, Joshua J.; Walsh, Michael P.; Wong, Sallene; Seto, Donald; Dyer, David W.; Chodosh, James; Jones, Morris S.

    2013-01-01

    ABSTRACT For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses. PMID:23572555

  4. Adenovirus DNA Replication

    PubMed Central

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease. PMID:23388625

  5. A human type 5 adenovirus-based tuberculosis vaccine induces robust T cell responses in humans despite preexisting anti-adenovirus immunity.

    PubMed

    Smaill, Fiona; Jeyanathan, Mangalakumari; Smieja, Marek; Medina, Maria Fe; Thanthrige-Don, Niroshan; Zganiacz, Anna; Yin, Cindy; Heriazon, Armando; Damjanovic, Daniela; Puri, Laura; Hamid, Jemila; Xie, Feng; Foley, Ronan; Bramson, Jonathan; Gauldie, Jack; Xing, Zhou

    2013-10-01

    There is an urgent need to develop new tuberculosis (TB) vaccines to safely and effectively boost Bacille Calmette-Guérin (BCG)-triggered T cell immunity in humans. AdHu5Ag85A is a recombinant human type 5 adenovirus (AdHu5)-based TB vaccine with demonstrated efficacy in a number of animal species, yet it remains to be translated to human applications. In this phase 1 study, we evaluated the safety and immunogenicity of AdHu5Ag85A in both BCG-naïve and previously BCG-immunized healthy adults. Intramuscular immunization of AdHu5Ag85A was safe and well tolerated in both trial volunteer groups. Moreover, although AdHu5Ag85A was immunogenic in both trial volunteer groups, it much more potently boosted polyfunctional CD4(+) and CD8(+) T cell immunity in previously BCG-vaccinated volunteers. Furthermore, despite prevalent preexisting anti-AdHu5 humoral immunity in most of the trial volunteers, we found little evidence that such preexisting anti-AdHu5 immunity significantly dampened the potency of AdHu5Ag85A vaccine. This study supports further clinical investigations of the AdHu5Ag85A vaccine for human applications. It also suggests that the widely perceived negative effect of preexisting anti-AdHu5 immunity may not be universally applied to all AdHu5-based vaccines against different types of human pathogens.

  6. 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

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

    PubMed

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

    2010-07-01

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

  8. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  9. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV.

  10. NY-ESO-1 specific antibody and cellular responses in melanoma patients primed with NY-ESO-1 protein in ISCOMATRIX and boosted with recombinant NY-ESO-1 fowlpox virus.

    PubMed

    Chen, Ji-Li; Dawoodji, Amina; Tarlton, Andrea; Gnjatic, Sacha; Tajar, Abdelouahid; Karydis, Ioannis; Browning, Judy; Pratap, Sarah; Verfaille, Christian; Venhaus, Ralph R; Pan, Linda; Altman, Douglas G; Cebon, Jonathan S; Old, Lloyd L; Nathan, Paul; Ottensmeier, Christian; Middleton, Mark; Cerundolo, Vincenzo

    2015-03-15

    Vaccination strategies based on repeated injections of NY-ESO-1 protein formulated in ISCOMATRIX particles (NY-ESO-1 ISCOMATRIX) have shown to elicit combined NY-ESO-1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime-boost strategies based on the combination with NY-ESO-1 ISCOMATRIX with different NY-ESO-1 boosting reagents could be used to increase NY-ESO-1 CD8(+) or CD4(+) T cell responses. To address this question, we carried out a randomized clinical trial in 39 high-risk, resected melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY-ESO-1 (rF-NY-ESO-1) (Arm A) or NY-ESO-1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY-ESO-1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY-ESO-1 ISCOMATRIX alone elicited a strong NY-ESO-1 specific CD4(+) T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8(+) T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF-NY-ESO-1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8(+) T cell responses. In addition, our results clearly identified immunodominant regions in the NY-ESO-1 protein: NY-ESO-179-102 and NY-ESO-1115-138 for CD4+ T cells and NY-ESO-185-108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY-ESO-1 protein should be considered in future clinical trials as immunodominant epitopes.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    2014-01-01

    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

  13. 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

  14. Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP(+)-dependent xylitol dehydrogenase.

    PubMed

    Khattab, Sadat Mohammad Rezq; Saimura, Masayuki; Kodaki, Tsutomu

    2013-06-10

    The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production.

  15. A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

    PubMed Central

    Ramos, Henrique Roman; Junqueira-de-Azevedo, Inácio de Loiola M.; Novo, Juliana Branco; Castro, Karen; Duarte, Clara Guerra; Machado-de-Ávila, Ricardo A.; Chavez-Olortegui, Carlos; Ho, Paulo Lee

    2016-01-01

    Background Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. Methods and Findings In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. Conclusion Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity. PMID:26938217

  16. A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis.

    PubMed

    Carson, Connor; Antoniou, Maria; Ruiz-Argüello, Maria Begoña; Alcami, Antonio; Christodoulou, Vasiliki; Messaritakis, Ippokratis; Blackwell, Jenefer M; Courtenay, Orin

    2009-02-11

    Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) and Leishmania homologue of the mammalian receptor for activated C kinase (LACK) against Leishmania major challenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host of Leishmania infantum, followed-up for 4 months post-vaccination. In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000 microg (high dose) or 100 microg (low dose) DNA prime (day 0) and 1x10(8)pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-gamma than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge with Leishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positive in vivo intradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-gamma in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/T(reg) response, as expected in the absence of in vivo restimulation or natural/experimental challenge with Leishmania. These collective results indicate significant antigen-specific type-1 responses and in vivo memory phase cellular immune

  17. Delivery of avian cytokines by adenovirus vectors.

    PubMed

    Johnson, M A; Pooley, C; Lowenthal, J W

    2000-01-01

    A fowl adenovirus serotype 8 (FAV-8) recombinant was constructed by inserting an expression cassette consisting of the FAV major late promoter/splice leader sequences (MLP/SL), the chicken interferon-gamma (ChIFN-gamma) gene and SV40 polyA into sites in the right hand end of the FAV-8 genome. One recombinant (A3-13) was constructed by an insertion of ChIFN-gamma into a 1.3 kilobase pair (kbp) deletion which removed a putative open reading frame (ORF) with identity to the CELO (FAV serotype 1) 36 kDa homologue. A second recombinant (S4) removed a further 0.9 kbp and a third recombinant (AA1) was constructed in a small 50 base pair (bp) SpeI deletion. The recombinants displayed differing growth characteristics in CK monolayers. A3-13 grew slowly and only attained a titre of 10(5) pfu/ml, S4 had intermediate growth and AA1 showed wild type growth kinetics. These differing growth properties indicated that removal of the 36 kDa homologue had an effect on growth in vitro. Supernatants from CK monolayers infected with the recombinant virus were assayed for the production of ChIFN-gamma. Detectable levels of ChIFN-gamma were observed in supernatants as early as 24 h post infection (p.i.), peaked at 48 h p.i. and this level was maintained for at least 10 days. The level of production of ChIFN-gamma correlated with each recombinant's growth characteristics in vitro. Chickens treated with rFAV-ChIFN-gamma showed increased weight gains compared to controls and suffered reduced weight loss when challenged with the coccidial parasite Eimeria acervulina. PMID:10717297

  18. Prime-Boost Vaccination with Toxoplasma Lysate Antigen, but Not with a Mixture of Recombinant Protein Antigens, Leads to Reduction of Brain Cyst Formation in BALB/c Mice

    PubMed Central

    Wagner, Angelika; Schabussova, Irma; Ruttkowski, Bärbel; Peschke, Roman; Kur, Józef; Kundi, Michael; Joachim, Anja; Wiedermann, Ursula

    2015-01-01

    Introduction Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking. Methods Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice. Results Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii. Conclusion In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further

  19. Adenovirus serotype 30 fiber does not mediate transduction via the coxsackie-adenovirus receptor.

    PubMed

    Law, Lane K; Davidson, Beverly L

    2002-01-01

    Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR. PMID:11752156

  20. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

  1. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses. PMID:27029122

  2. Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

    SciTech Connect

    Park, Ki Seok; Lee, Jiyeung; Ahn, So Shin; Byun, Young-Ho; Seong, Baik Lin; Baek, Yun Hee; Song, Min-Suk; Choi, Young Ki; Na, Yun Jeong; Hwang, Inhwan; Sung, Young Chul; Lee, Chang Geun

    2009-12-20

    Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8{sup +} T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.

  3. Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

    PubMed

    Widjojoatmodjo, Myra N; Bogaert, Lies; Meek, Bob; Zahn, Roland; Vellinga, Jort; Custers, Jerome; Serroyen, Jan; Radošević, Katarina; Schuitemaker, Hanneke

    2015-10-01

    RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

  4. Severe conjunctivitis due to multidrug-resistant Neisseria gonorrhoeae and adenovirus 53 coinfection in a traveler returning from Thailand.

    PubMed

    Tappe, Dennis; Mueller, Andreas; Weißbrich, Benedikt; Schubert, Jörg; Schargus, Marc; Stich, August

    2013-01-01

    A male traveler returning from Thailand with severe bilateral conjunctivitis was tested for causative pathogens by culture and polymerase chain reaction in late 2010. The culturally grown Neisseria gonorrhoeae strain was resistant against penicillin, ciprofloxacin, and tetracycline. The patient was also found to have an eye infection with the unusual and likely recombinant adenovirus type 53. Besides multidrug-resistant gonococcal strains the unusual adenovirus strain is found circulating in Asia and both pathogens may be a risk for travelers.

  5. Construction of mouse adenovirus type 1 mutants.

    PubMed

    Cauthen, Angela N; Welton, Amanda R; Spindler, Katherine R

    2007-01-01

    Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wild-type mouse adenovirus, production of viral DNA and viral DNA-protein complex, and transfection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of the mutant viruses are also presented.

  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. 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

  8. 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

  9. Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

    PubMed

    Thorne, Barbara A; Quigley, Paulene; Nichols, Gina; Moore, Christine; Pastor, Eric; Price, David; Ament, Jon W; Takeya, Ryan K; Peluso, Richard W

    2008-01-01

    Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Human adenoviruses: propagation, purification, quantification, and storage.

    PubMed

    Green, Maurice; Loewenstein, Paul M

    2006-01-01

    Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

  13. Protective immunity against a lethal respiratory Yersinia pestis challenge induced by V antigen or the F1 capsular antigen incorporated into adenovirus capsid.

    PubMed

    Boyer, Julie L; Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Chiuchiolo, Maria J; Senina, Svetlana; Perlin, David; Crystal, Ronald G

    2010-07-01

    The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective

  14. 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

  15. 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.

  16. A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

    PubMed

    Sha, Jian; Kirtley, Michelle L; Klages, Curtis; Erova, Tatiana E; Telepnev, Maxim; Ponnusamy, Duraisamy; Fitts, Eric C; Baze, Wallace B; Sivasubramani, Satheesh K; Lawrence, William S; Patrikeev, Igor; Peel, Jennifer E; Andersson, Jourdan A; Kozlova, Elena V; Tiner, Bethany L; Peterson, Johnny W; McWilliams, David; Patel, Snehal; Rothe, Eric; Motin, Vladimir L; Chopra, Ashok K

    2016-07-01

    Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. PMID:27170642

  17. A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

    PubMed

    Sha, Jian; Kirtley, Michelle L; Klages, Curtis; Erova, Tatiana E; Telepnev, Maxim; Ponnusamy, Duraisamy; Fitts, Eric C; Baze, Wallace B; Sivasubramani, Satheesh K; Lawrence, William S; Patrikeev, Igor; Peel, Jennifer E; Andersson, Jourdan A; Kozlova, Elena V; Tiner, Bethany L; Peterson, Johnny W; McWilliams, David; Patel, Snehal; Rothe, Eric; Motin, Vladimir L; Chopra, Ashok K

    2016-07-01

    Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

  18. Analysis of purified Wild type and mutant adenovirus particles by SILAC based quantitative proteomics

    PubMed Central

    Alqahtani, Ali; Heesom, Kate; Bramson, Jonathan L.; Curiel, David; Ugai, Hideyo

    2014-01-01

    We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120. PMID:25096814

  19. Viable adenovirus vaccine prototypes: high-level production of a papillomavirus capsid antigen from the major late transcriptional unit.

    PubMed

    Berg, Michael; Difatta, Julie; Hoiczyk, Egbert; Schlegel, Richard; Ketner, Gary

    2005-03-22

    Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection. Vaccination with virus-like particles (VLPs) composed of recombinant HPV L1 completely prevents persistent HPV infection [Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M. & Jansen, K. U. (2002) N. Engl. J. Med. 347, 1645-1651], suggesting that L1 expressed from recombinant adenoviruses might provide protective immunity. In our recombinants, COPV L1 is incorporated into adenovirus late region 5 (Ad L5) and is expressed as a member of the adenoviral major late transcriptional unit (MLTU). COPV L1 production by the most prolific recombinant is comparable to that of the most abundant adenoviral protein, hexon. COPV L1 production by recombinants is influenced by Ad L5 gene order, the specific mRNA processing signals associated with COPV L1, and the state of a putative splicing inhibitor in the COPV L1 gene. Recombinant COPV L1 protein assembles into VLPs that react with an antibody specific for conformational epitopes on native COPV L1 protein that correlate with protection in vivo. The designs of these recombinants can be applied directly to the production of recombinants appropriate for assessing immunogenicity and protective efficacy in animal models and in human trials. PMID:15767581

  20. Viable adenovirus vaccine prototypes: High-level production of a papillomavirus capsid antigen from the major late transcriptional unit

    PubMed Central

    Berg, Michael; DiFatta, Julie; Hoiczyk, Egbert; Schlegel, Richard; Ketner, Gary

    2005-01-01

    Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection. Vaccination with virus-like particles (VLPs) composed of recombinant HPV L1 completely prevents persistent HPV infection [Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M. & Jansen, K. U. (2002) N. Engl. J. Med. 347, 1645–1651], suggesting that L1 expressed from recombinant adenoviruses might provide protective immunity. In our recombinants, COPV L1 is incorporated into adenovirus late region 5 (Ad L5) and is expressed as a member of the adenoviral major late transcriptional unit (MLTU). COPV L1 production by the most prolific recombinant is comparable to that of the most abundant adenoviral protein, hexon. COPV L1 production by recombinants is influenced by Ad L5 gene order, the specific mRNA processing signals associated with COPV L1, and the state of a putative splicing inhibitor in the COPV L1 gene. Recombinant COPV L1 protein assembles into VLPs that react with an antibody specific for conformational epitopes on native COPV L1 protein that correlate with protection in vivo. The designs of these recombinants can be applied directly to the production of recombinants appropriate for assessing immunogenicity and protective efficacy in animal models and in human trials. PMID:15767581

  1. Safety and Immunogenicity Study of Multiclade HIV-1 Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA Vaccine in Africa

    PubMed Central

    Allen, Susan; Than, Soe; Adams, Elizabeth M.; Graham, Barney S.; Koup, Richard A.; Bailer, Robert T.; Smith, Carol; Dally, Len; Tarragona-Fiol, Tony; Bergin, Philip J.; Hayes, Peter; Ho, Martin; Loughran, Kelley; Komaroff, Wendy; Stevens, Gwynneth; Thomson, Helen; Boaz, Mark J.; Cox, Josephine H.; Schmidt, Claudia; Gilmour, Jill; Nabel, Gary J.; Fast, Patricia

    2010-01-01

    Background We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults. Methodology/Principal Findings Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone. Conclusions/Significance The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints. Trial

  2. Performance Boosting Additive

    NASA Technical Reports Server (NTRS)

    1999-01-01

    Mainstream Engineering Corporation was awarded Phase I and Phase II contracts from Goddard Space Flight Center's Small Business Innovation Research (SBIR) program in early 1990. With support from the SBIR program, Mainstream Engineering Corporation has developed a unique low cost additive, QwikBoost (TM), that increases the performance of air conditioners, heat pumps, refrigerators, and freezers. Because of the energy and environmental benefits of QwikBoost, Mainstream received the Tibbetts Award at a White House Ceremony on October 16, 1997. QwikBoost was introduced at the 1998 International Air Conditioning, Heating, and Refrigeration Exposition. QwikBoost is packaged in a handy 3-ounce can (pressurized with R-134a) and will be available for automotive air conditioning systems in summer 1998.

  3. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  4. Adenovirus Early Proteins and Host Sumoylation.

    PubMed

    Sohn, Sook-Young; Hearing, Patrick

    2016-01-01

    The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  5. 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

  6. Priming with a Simplified Intradermal HIV-1 DNA Vaccine Regimen followed by Boosting with Recombinant HIV-1 MVA Vaccine Is Safe and Immunogenic: A Phase IIa Randomized Clinical Trial

    PubMed Central

    Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L.; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

    2015-01-01

    Background Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. Methods In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two “simplified” regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. Results 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. Conclusions A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. Trial Registration World Health

  7. A novel adenovirus vector for easy cloning in the E3 region downstream of the CMV promoter.

    PubMed

    Mailly, Laurent; Boulade-Ladame, Charlotte; Orfanoudakis, Georges; Deryckere, François

    2008-01-01

    The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells. PMID:18538014

  8. Acid-Soluble Material of Adenovirus

    PubMed Central

    Boulanger, P. A.; Jaume, F.; Flamencourt, P.; Biserte, G.

    1970-01-01

    Two methods are described for adenovirus capsid disruption and extraction of acid-soluble proteins from the viral core. The acid-soluble material of adenovirus consisted of three major proteins, one of them being selectively extracted after mild disruption of the virus particle. Some chemical properties of these proteins are reported. Images PMID:4986288

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

    PubMed

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

    2015-08-12

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

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

    PubMed

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

    2015-08-12

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

  11. 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

  12. 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

  13. Online Bagging and Boosting

    NASA Technical Reports Server (NTRS)

    Oza, Nikunji C.

    2005-01-01

    Bagging and boosting are two of the most well-known ensemble learning methods due to their theoretical performance guarantees and strong experimental results. However, these algorithms have been used mainly in batch mode, i.e., they require the entire training set to be available at once and, in some cases, require random access to the data. In this paper, we present online versions of bagging and boosting that require only one pass through the training data. We build on previously presented work by presenting some theoretical results. We also compare the online and batch algorithms experimentally in terms of accuracy and running time.

  14. 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

  15. 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.

  16. Clinical and Virologic Characteristics May Aid Distinction of Acute Adenovirus Disease from Kawasaki Disease with Incidental Adenovirus Detection.

    PubMed

    Song, Eunkyung; Kajon, Adriana E; Wang, Huanyu; Salamon, Doug; Texter, Karen; Ramilo, Octavio; Leber, Amy; Jaggi, Preeti

    2016-03-01

    Incidental adenovirus detection in Kawasaki disease (KD) is important to differentiate from acute adenovirus disease. Twenty-four of 25 children with adenovirus disease and mimicking features of KD had <4 KD-like features, predominance of species B or E, and higher viral burden compared with those with KD and incidental adenovirus detection. PMID:26707621

  17. 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

  18. Octane boosting catalyst

    SciTech Connect

    Miller, J.G.; Pellet, R.J.; Shamshoun, E.S.; Rabo, J.A

    1989-02-07

    The invention provides petroleum cracking and octane boosting catalysts containing a composite of an intermediate pore NZMS in combination with another non-zeolitic molecular sieve having the same framework structure, and processes for cracking of petroleum for the purpose of enhancing the octane rating of the gasoline produced.

  19. 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

  20. 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.

  1. Strong HIV-specific CD4+ and CD8+ T-lymphocyte proliferative responses in healthy individuals immunized with an HIV-1 DNA vaccine and boosted with recombinant modified vaccinia virus ankara expressing HIV-1 genes.

    PubMed

    Aboud, Said; Nilsson, Charlotta; Karlén, Katarina; Marovich, Mary; Wahren, Britta; Sandström, Eric; Gaines, Hans; Biberfeld, Gunnel; Godoy-Ramirez, Karina

    2010-07-01

    We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag, env, rev, and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag, env, and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [(3)H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed (n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [(3)H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4(+) T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8(+) T-cell responses. There was strong correlation between the proliferative responses measured by the [(3)H]thymidine uptake assay and CD4(+) T-cell FASCIA-WB (r = 0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4(+) T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8(+) T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [(3)H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [(3)H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

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

    PubMed

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

    2015-07-17

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

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

    PubMed Central

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

    2015-01-01

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

  4. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  5. A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

    PubMed Central

    Pereira, Isabela Resende; Vilar-Pereira, Glaucia; Marques, Virgínia; da Silva, Andrea Alice; Caetano, Bráulia; Moreira, Otacilio Cruz; Machado, Alexandre Vieira; Bruna-Romero, Oscar; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the

  6. Infectious entry pathway of adenovirus type 2.

    PubMed Central

    Varga, M J; Weibull, C; Everitt, E

    1991-01-01

    Internalization of the infectious fraction of human adenovirus type 2 into HeLa cells was followed by a quantitative internalization assay. Treatments known to selectively block receptor-mediated endocytosis reduced the internalization of infectious virus to an extent close to the reduction of endocytosis of transferrin. This suggests that one of the first steps in the infectious cycle of adenovirus type 2 is internalization by the coated-pit and -vesicle pathway. Images PMID:1920625

  7. 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.

  8. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses. PMID:14654922

  9. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

  10. Adenovirus-vectored Ebola vaccines.

    PubMed

    Gilbert, Sarah C

    2015-01-01

    The 2014 outbreak of Ebola virus disease in West Africa has highlighted the need for the availability of effective vaccines against outbreak pathogens that are suitable for use in frontline workers who risk their own health in the course of caring for those with the disease, and also for members of the community in the affected area. Along with effective contact tracing and quarantine, use of a vaccine as soon as an outbreak is identified could greatly facilitate rapid control and prevent the outbreak from spreading. This review describes the progress that has been made in producing and testing adenovirus-based Ebola vaccines in both pre-clinical and clinical studies, and considers the likely future use of these vaccines.

  11. 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.

  12. Parenteral adenoviral boost enhances BCG induced protection, but not long term survival in a murine model of bovine TB.

    PubMed

    Kaveh, Daryan A; Garcia-Pelayo, M Carmen; Webb, Paul R; Wooff, Esen E; Bachy, Véronique S; Hogarth, Philip J

    2016-07-25

    Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.4 (BCG/Ad-TB10.4), we demonstrate, parenteral boost of BCG immunised mice to induce specific CD8(+) IFN-γ producing T cells via synergistic priming of new epitopes. This induces significant improvement in pulmonary protection against Mycobacterium bovis over that provided by BCG when assessed in a standard 4week challenge model. However, in a stringent, year-long survival study, BCG/Ad-TB10.4 did not improve outcome over BCG, which we suggest may be due to the lack of additional memory cells (IL-2(+)) induced by boosting. These data indicate BCG-prime/parenteral-Ad-TB10.4-boost to be a promising candidate, but also highlight the need for further understanding of the mechanisms of T cell priming and associated memory using Ad delivery systems. That we were able to generate significant improvement in pulmonary protection above BCG with parenteral, rather than mucosal administration of boost vaccine is critical; suggesting that the generation of effective mucosal immunity is possible, without the risks and challenges of mucosal administration, but that further work to specifically enhance sustained protective immunity is required.

  13. Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice.

    PubMed

    van Heeckeren, A; Ferkol, T; Tosi, M

    1998-03-01

    Cystic fibrosis (CF) patients have endobronchial inflammation caused by infection with mucoid Pseudomonas aeruginosa. Since adenovirus vectors are being studied for gene therapy for CF, we sought to determine whether bronchopulmonary inflammation would influence adenovirus-mediated gene transfer. We hypothesized that bronchopulmonary inflammation in mice inoculated with mucoid P. aeruginosa would be associated with a decrease in the efficacy of adenovirus-mediated gene transfer. Agarose beads embedded with mucoid P. aeruginosa (6 x 10(4) c.f.u. per mouse) were inoculated transtracheally into C57BL/6 mice. Control mice received sterile agarose beads. Ten days after inoculation with agarose beads, recombinant adenovirus containing the beta-galactosidase reporter gene (Ad2/beta Gal-2) was administered intranasally (1.1 x 10(9) IU per mouse), and mice were killed 3 days later. The extent of inflammation, determined by neutrophil numbers in bronchoalveolar lavage fluid and by areal lung inflammation, was significantly greater in mice inoculated with P. aeruginosa-laden agarose beads and Ad2/beta Gal-2 compared with controls. Mice that had received Pseudomonas-laden agarose beads and Ad2/beta Gal-2 had significantly fewer (P < 0.015) airway epithelial cells transduced (4.1 +/- 0.9%) compared with mice that received sterile agarose beads and Ad2/beta Gal-2 (9.4 +/- 1.4%). These results indicate that the efficacy of adenovirus-mediated gene transfer is reduced in Pseudomonas-induced bronchopulmonary inflammation.

  14. Molecular epidemiology of subgenus F adenoviruses associated with pediatric gastroenteritis during eight years in Hiroshima Prefecture as a limited area.

    PubMed

    Fukuda, S; Kuwayama, M; Takao, S; Shimazu, Y; Miyazaki, K

    2006-12-01

    We have studied the prevalence of the subgenus F adenoviruses and the molecular characteristics of adenovirus type 41 in Hiroshima Prefecture, Japan, as a limited area during the period of 1997-2004. Subgenus F adenoviruses were detected in 30 (3.4%) of 892 fecal specimens by enzyme immunoassay (EIA), and 80.0% (24 of 30) of positive patients were <36 months old. One (3.3%) and 29 (96.7%) of the 30 EIA-positive specimens were adenoviruses type 40 (Ad40) and 41 (Ad41), respectively. The genomes of Ad41 strains amplified by PCR were divided into two genomic type clusters (GTC1 and GTC2) based on the hexon gene as described by Li et al. (J Clin Microbiol 42: 4032-4039, 2004.). Twenty-one (95.5%) of 22 Ad41 strains detected between 2000 and 2004 belonged to GTC1, whereas all seven strains detected between 1997 and 1999 belonged to GTC2. These genomic typings were the same for the hexon and fiber genes except for one strain. This strain contained a hexon gene belonging to GTC1 and a fiber gene belonging to GTC2 and was considered to be a recombinant between adenoviruses of these types. PMID:16847553

  15. Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

    PubMed Central

    Liebermann, Herbert; Mentel, Renate; Bauer, Ulrike; Pring-Åkerblom, Patricia; Dölling, Rudolf; Modrow, Susanne; Seidel, Werner

    1998-01-01

    The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells. PMID:9765458

  16. human adenoviruses role in ophthalmic pterygium formation

    PubMed Central

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

    2015-01-01

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

  17. Gradient boosting machines, a tutorial

    PubMed Central

    Natekin, Alexey; Knoll, Alois

    2013-01-01

    Gradient boosting machines are a family of powerful machine-learning techniques that have shown considerable success in a wide range of practical applications. They are highly customizable to the particular needs of the application, like being learned with respect to different loss functions. This article gives a tutorial introduction into the methodology of gradient boosting methods with a strong focus on machine learning aspects of modeling. A theoretical information is complemented with descriptive examples and illustrations which cover all the stages of the gradient boosting model design. Considerations on handling the model complexity are discussed. Three practical examples of gradient boosting applications are presented and comprehensively analyzed. PMID:24409142

  18. Analytic boosted boson discrimination

    NASA Astrophysics Data System (ADS)

    Larkoski, Andrew J.; Moult, Ian; Neill, Duff

    2016-05-01

    Observables which discriminate boosted topologies from massive QCD jets are of great importance for the success of the jet substructure program at the Large Hadron Collider. Such observables, while both widely and successfully used, have been studied almost exclusively with Monte Carlo simulations. In this paper we present the first all-orders factorization theorem for a two-prong discriminant based on a jet shape variable, D 2, valid for both signal and background jets. Our factorization theorem simultaneously describes the production of both collinear and soft subjets, and we introduce a novel zero-bin procedure to correctly describe the transition region between these limits. By proving an all orders factorization theorem, we enable a systematically improvable description, and allow for precision comparisons between data, Monte Carlo, and first principles QCD calculations for jet substructure observables. Using our factorization theorem, we present numerical results for the discrimination of a boosted Z boson from massive QCD background jets. We compare our results with Monte Carlo predictions which allows for a detailed understanding of the extent to which these generators accurately describe the formation of two-prong QCD jets, and informs their usage in substructure analyses. Our calculation also provides considerable insight into the discrimination power and calculability of jet substructure observables in general.

  19. Analytic boosted boson discrimination

    DOE PAGESBeta

    Larkoski, Andrew J.; Moult, Ian; Neill, Duff

    2016-05-20

    Observables which discriminate boosted topologies from massive QCD jets are of great importance for the success of the jet substructure program at the Large Hadron Collider. Such observables, while both widely and successfully used, have been studied almost exclusively with Monte Carlo simulations. In this paper we present the first all-orders factorization theorem for a two-prong discriminant based on a jet shape variable, D2, valid for both signal and background jets. Our factorization theorem simultaneously describes the production of both collinear and soft subjets, and we introduce a novel zero-bin procedure to correctly describe the transition region between these limits.more » By proving an all orders factorization theorem, we enable a systematically improvable description, and allow for precision comparisons between data, Monte Carlo, and first principles QCD calculations for jet substructure observables. Using our factorization theorem, we present numerical results for the discrimination of a boosted Z boson from massive QCD background jets. We compare our results with Monte Carlo predictions which allows for a detailed understanding of the extent to which these generators accurately describe the formation of two-prong QCD jets, and informs their usage in substructure analyses. In conclusion, our calculation also provides considerable insight into the discrimination power and calculability of jet substructure observables in general.« less

  20. 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 ...

  1. Components of Adenovirus Genome Packaging

    PubMed Central

    Ahi, Yadvinder S.; Mittal, Suresh K.

    2016-01-01

    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

  2. ADV36 adipogenic adenovirus in human liver disease

    PubMed Central

    Trovato, Francesca M; Catalano, Daniela; Garozzo, Adriana; Martines, G Fabio; Pirri, Clara; Trovato, Guglielmo M

    2014-01-01

    Obesity and liver steatosis are usually described as related diseases. Obesity is regarded as exclusive consequence of an imbalance between food intake and physical exercise, modulated by endocrine and genetic factors. Non-alcoholic fatty liver disease (NAFLD), is a condition whose natural history is related to, but not completely explained by over-nutrition, obesity and insulin resistance. There is evidence that environmental infections, and notably adipogenic adenoviruses (ADV) infections in humans, are associated not only with obesity, which is sufficiently established, but also with allied conditions, such as fatty liver. In order to elucidate the role, if any, of previous ADV36 infection in humans, we investigated association of ADV36-ADV37 seropositivity with obesity and fatty liver in humans. Moreover, the possibility that lifestyle-nutritional intervention in patients with NAFLD and different ADV36 seropositive status, achieves different clinical outcomes on ultrasound bright liver imaging, insulin resistance and obesity was challenged. ADV36 seropositive patients have a more consistent decrease in insulin resistance, fatty liver severity and body weight in comparison with ADV36 seronegative patients, indicating a greater responsiveness to nutritional intervention. These effects were not dependent on a greater pre-interventional body weight and older age. These results imply that no obvious disadvantage - and, seemingly, that some benefit - is linked to ADV36 seropositivity, at least in NAFLD. ADV36 previous infection can boost weight loss and recovery of insulin sensitivity under interventional treatment. PMID:25356033

  3. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  4. The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3.

    PubMed

    Lapointe, J M; Hedges, J F; Woods, L W; Reubel, G H; MacLachlan, N J

    1999-01-01

    DNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct.

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

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

    2007-01-01

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

  7. 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.

  8. 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

  9. Bidirectional buck boost converter

    DOEpatents

    Esser, Albert Andreas Maria

    1998-03-31

    A bidirectional buck boost converter and method of operating the same allows regulation of power flow between first and second voltage sources in which the voltage level at each source is subject to change and power flow is independent of relative voltage levels. In one embodiment, the converter is designed for hard switching while another embodiment implements soft switching of the switching devices. In both embodiments, first and second switching devices are serially coupled between a relatively positive terminal and a relatively negative terminal of a first voltage source with third and fourth switching devices serially coupled between a relatively positive terminal and a relatively negative terminal of a second voltage source. A free-wheeling diode is coupled, respectively, in parallel opposition with respective ones of the switching devices. An inductor is coupled between a junction of the first and second switching devices and a junction of the third and fourth switching devices. Gating pulses supplied by a gating circuit selectively enable operation of the switching devices for transferring power between the voltage sources. In the second embodiment, each switching device is shunted by a capacitor and the switching devices are operated when voltage across the device is substantially zero.

  10. Bidirectional buck boost converter

    DOEpatents

    Esser, A.A.M.

    1998-03-31

    A bidirectional buck boost converter and method of operating the same allows regulation of power flow between first and second voltage sources in which the voltage level at each source is subject to change and power flow is independent of relative voltage levels. In one embodiment, the converter is designed for hard switching while another embodiment implements soft switching of the switching devices. In both embodiments, first and second switching devices are serially coupled between a relatively positive terminal and a relatively negative terminal of a first voltage source with third and fourth switching devices serially coupled between a relatively positive terminal and a relatively negative terminal of a second voltage source. A free-wheeling diode is coupled, respectively, in parallel opposition with respective ones of the switching devices. An inductor is coupled between a junction of the first and second switching devices and a junction of the third and fourth switching devices. Gating pulses supplied by a gating circuit selectively enable operation of the switching devices for transferring power between the voltage sources. In the second embodiment, each switching device is shunted by a capacitor and the switching devices are operated when voltage across the device is substantially zero. 20 figs.

  11. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells*

    PubMed Central

    Luo, Xu-wei; Liu, Kang; Chen, Zhu; Zhao, Ming; Han, Xiao-wei; Bai, Yi-guang; Feng, Gang

    2016-01-01

    Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells. PMID:26739524

  12. Optimization of Prime-Boost Vaccination Strategies Against Mouse-Adapted Ebolavirus in a Short-Term Protection Study.

    PubMed

    Aviles, Jenna; Bello, Alexander; Wong, Gary; Fausther-Bovendo, Hugues; Qiu, Xiangguo; Kobinger, Gary

    2015-10-01

    In nonhuman primates, complete protection against an Ebola virus (EBOV) challenge has previously been achieved after a single injection with several vaccine platforms. However, long-term protection against EBOV after a single immunization has not been demonstrated to this date. Interestingly, prime-boost regimens have demonstrated longer protection against EBOV challenge, compared with single immunizations. Since prime-boost regimens have the potential to achieve long-term protection, determining optimal vector combinations is crucial. However, testing prime-boost efficiency in long-term protection studies is time consuming and resource demanding. Here, we investigated the optimal prime-boost combination, using DNA, porcine-derived adeno-associated virus serotype 6 (AAV-po6), and human adenovirus serotype 5 (Ad5) vector, in a short-term protection study in the mouse model of EBOV infection. In addition, we also investigated which immune parameters were indicative of a strong boost. Each vaccine platform was titrated in mice to identify which dose (single immunization) induced approximately 20% protection after challenge with a mouse-adapted EBOV. These doses were then used to determine the protection efficacy of various prime-boost combinations, using the same mouse model. In addition, humoral and cellular immune responses against EBOV glycoprotein were analyzed by an enzyme-linked immunosorbent assay, a neutralizing antibody assay, and an interferon γ-specific enzyme-linked immunospot assay. When DNA was used as a prime, Ad5 boost induced the best protection, which correlated with a higher cellular response. In contrast, when AAV-po6 or Ad5 were injected first, better protection was achieved after DNA boost, and this correlated with a higher total glycoprotein-specific immunoglobulin G titer. Prime-boost regimens using independent vaccine platforms may provide a useful strategy to induce long-term immune protection against filoviruses.

  13. Optimization of Prime-Boost Vaccination Strategies Against Mouse-Adapted Ebolavirus in a Short-Term Protection Study.

    PubMed

    Aviles, Jenna; Bello, Alexander; Wong, Gary; Fausther-Bovendo, Hugues; Qiu, Xiangguo; Kobinger, Gary

    2015-10-01

    In nonhuman primates, complete protection against an Ebola virus (EBOV) challenge has previously been achieved after a single injection with several vaccine platforms. However, long-term protection against EBOV after a single immunization has not been demonstrated to this date. Interestingly, prime-boost regimens have demonstrated longer protection against EBOV challenge, compared with single immunizations. Since prime-boost regimens have the potential to achieve long-term protection, determining optimal vector combinations is crucial. However, testing prime-boost efficiency in long-term protection studies is time consuming and resource demanding. Here, we investigated the optimal prime-boost combination, using DNA, porcine-derived adeno-associated virus serotype 6 (AAV-po6), and human adenovirus serotype 5 (Ad5) vector, in a short-term protection study in the mouse model of EBOV infection. In addition, we also investigated which immune parameters were indicative of a strong boost. Each vaccine platform was titrated in mice to identify which dose (single immunization) induced approximately 20% protection after challenge with a mouse-adapted EBOV. These doses were then used to determine the protection efficacy of various prime-boost combinations, using the same mouse model. In addition, humoral and cellular immune responses against EBOV glycoprotein were analyzed by an enzyme-linked immunosorbent assay, a neutralizing antibody assay, and an interferon γ-specific enzyme-linked immunospot assay. When DNA was used as a prime, Ad5 boost induced the best protection, which correlated with a higher cellular response. In contrast, when AAV-po6 or Ad5 were injected first, better protection was achieved after DNA boost, and this correlated with a higher total glycoprotein-specific immunoglobulin G titer. Prime-boost regimens using independent vaccine platforms may provide a useful strategy to induce long-term immune protection against filoviruses. PMID:26038398

  14. 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

  15. Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond.

    PubMed

    Kaufmann, Johanna K; Nettelbeck, Dirk M

    2012-07-01

    Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.

  16. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

    PubMed

    Imler, J L; Chartier, C; Dreyer, D; Dieterle, A; Sainte-Marie, M; Faure, T; Pavirani, A; Mehtali, M

    1996-01-01

    Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus. PMID:8929914

  17. Clinical protocol. Purging of autologous stem cell sources with bcl-x(s) adenovirus for women undergoing high-dose chemotherapy for stage IV breast carcinoma.

    PubMed

    Ayash, L J; Clarke, M; Adams, P; Ferrara, J; Ratanatharathorn, V; Reynolds, C; Roessler, B; Silver, S; Strawderman, M; Uberti, J; Wicha, M

    2001-11-01

    High-dose chemotherapy (HDCT) and autologous bone marrow transplantation (BMT) is frequently used to treat patients with metastatic cancer including breast cancer and neuroblastoma. However, the bone marrow of such patients is often contaminated with tumor cells. Recently, we have found that a recombinant adenovirus vector that contains a bcl-x, minigene (a dominant negative inhibitor of the bcl-2 family), called the bcl-x(s) adenovirus, is lethal to cancer cells derived from epithelial tissues, but not to normal human hematopoietic cells. To determine the mechanism, by which this virus spares normal hematopoietic cells, we isolated normal mouse hematopoietic stem cells and infected them with an adenovirus that contains a beta-galactosidase minigene. Such cells do not express beta-galactosidase, indicating that hematopoietic stem cells do not express transgene encoded by adenovirus vectors based upon the RSV-AD5 vector system. When breast cancer cells mixed with hematopoietic cells were infected with the bcl-x(s) adenovirus, cancer cells were selectively killed by the suicide adenoviruses. Hematopoietic cells exposed to the suicide vectors were able to reconstitute the bone marrow of mice exposed to lethal doses of y-irradiation. These studies suggest that adenovirus suicide vectors may provide a simple and effective method to selectively eliminate cancer cells derived from epithelial tissue that contaminate bone marrow to be used for autologous BMT. We therefore propose to initiate a phase I clinical trial to test the safety of this virus in women with breast cancer undergoing high does chemotherapy and autologous BMT.

  18. Isolation and Characterization of an Equine Adenovirus

    PubMed Central

    Ardans, Alexander A.; Pritchett, Randall F.; Zee, Yuan Chung

    1973-01-01

    A viral agent was isolated from lung tissue obtained upon necropsy of an Arabian foal which had exhibited clinical signs of pneumonia. The virus is 75 nm in diameter, cubic in symmetry, and resistant to chloroform and low pH (3.0). It contains deoxyribonucleic acid and has a buoyant density of 1.31 g/cm3 in cesium chloride. These findings indicate that the virus is a member of the adenovirus group. Images PMID:16558078

  19. 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).

  20. 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.

  1. 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

  2. Fiber-modified adenoviruses for targeted gene therapy.

    PubMed

    Wu, Hongju; Curiel, David T

    2008-01-01

    Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector. To achieve highly efficient and specific gene delivery, it is often necessary to re-direct Ad5 tropism. Because the capsid protein fiber plays an essential role in directing Ad5 infection, our laboratory attempted to re-target Ad5 through fiber modification. We have developed two strategies in this regard. One is a bi-specific adaptor protein strategy, in which the adaptor protein is designed to bind both the Ad5 fiber and an alternative cell-surface receptor. Another is genetic modification, in which alternative targeting motifs are genetically incorporated into the fiber knob domain so that the Ad5 vectors can infect cells through the alternative receptors. In this chapter, we will focus on the genetic fiber modification strategy and provide a detailed protocol for generation of fiber-modified Ad5 vectors. A series of techniques/procedures used in our laboratory will be described, which include the generation of fiber-modified Ad5 genome by homologous recombination in a bacterial system, rescuing the modified Ad5 viruses, virus amplification and purification, and virus titration.

  3. 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.

  4. 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)

  5. Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells

    PubMed Central

    Dmitriev, Igor; Kashentseva, Elena; Rogers, Buck E.; Krasnykh, Victor; Curiel, David T.

    2000-01-01

    Human adenovirus (Ad) is extensively used 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 target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency. PMID:10888627

  6. 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

  7. 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

  8. Prime-Boost Vaccination with rBCG/rAd35 Enhances CD8+ Cytolytic T-Cell Responses in Lesions from Mycobacterium Tuberculosis –Infected Primates

    PubMed Central

    Rahman, Sayma; Magalhaes, Isabelle; Rahman, Jubayer; Ahmed, Raija K; Sizemore, Donata R; Scanga, Charles A; Weichold, Frank; Verreck, Frank; Kondova, Ivanela; Sadoff, Jerry; Thorstensson, Rigmor; Spångberg, Mats; Svensson, Mattias; Andersson, Jan; Maeurer, Markus; Brighenti, Susanna

    2012-01-01

    To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8+ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3+ and CD8+ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8+ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals. PMID:22396020

  9. 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.

  10. 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

  11. A Novel Adenovirus in Chinstrap Penguins (Pygoscelis antarctica) in Antarctica

    PubMed Central

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won

    2014-01-01

    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins. PMID:24811321

  12. A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

    PubMed

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won

    2014-05-07

    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  13. Representing Arbitrary Boosts for Undergraduates.

    ERIC Educational Resources Information Center

    Frahm, Charles P.

    1979-01-01

    Presented is a derivation for the matrix representation of an arbitrary boost, a Lorentz transformation without rotation, suitable for undergraduate students with modest backgrounds in mathematics and relativity. The derivation uses standard vector and matrix techniques along with the well-known form for a special Lorentz transformation. (BT)

  14. Experimental adenovirus hemorrhagic disease in yearling black-tailed deer.

    PubMed

    Woods, L W; Hanley, R S; Chiu, P H; Burd, M; Nordhausen, R W; Stillian, M H; Swift, P K

    1997-10-01

    An apparently novel adenovirus was associated with an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California (USA) during 1993-1994. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. Six black-tailed yearling deer (O. hemionus columbianus) were inoculated with purified adenovirus isolated from a black-tailed fawn that died of acute adenovirus hemorrhagic disease during the epizootic. Three of six inoculated deer also received intramuscular injections of dexamethasone sodium phosphate every 3 days during the study. Eight days post-inoculation, one deer (without dexamethasone) developed bloody diarrhea and died. Necropsy and histopathologic findings were identical to lesions in free-ranging animals that died of the natural disease. Hemorrhagic enteropathy and pulmonary edema were the significant necropsy findings and there was microscopic vascular damage and endothelial intranuclear inclusion bodies in the vessels of the intestines and lungs. Adenovirus was identified in necrotic endothelial cells in the lungs by fluorescent antibody staining, immunohistochemistry and by transmission electron microscopy. Adenovirus was reisolated from tissues of the animal that died of experimental adenovirus hemorrhagic disease. Similar gross and microscopic lesions were absent in four of six adenovirus-inoculated deer and in the negative control animal which were necropsied at variable intervals during the 14 wk study. One deer was inoculated with purified adenovirus a second time, 12 wk after the first inoculation. Fifteen days after the second inoculation, this deer developed severe ulceration of the tongue, pharynx and rumen and necrotizing osteomyelitis of the mandible which was associated with

  15. Neutralization of adenoviruses: kinetics, stoichiometry, and mechanisms.

    PubMed Central

    Wohlfart, C

    1988-01-01

    Kinetic curves for neutralization of adenovirus type 2 with anti-hexon serum revealed no lag periods even when the serum was highly diluted or when the temperature was lowered to 4 degrees C, thus indicating a single-hit mechanism. Multiplicity curves determined with anti-hexon serum displayed a linear correlation between the degree of neutralization and dilution of antiserum. Neutralization values experimentally obtained under steady-state conditions fully fitted a single-hit model based on Poisson calculations. Quantitation of the amount of 125I-labeled type-specific anti-hexon antibodies needed for full neutralization of adenovirus showed that 1.4 antibodies were attached per virion under such conditions. Virions already attached to HeLa cells at 4 degrees C were, to a large extent, neutralizable by anti-hexon serum, whereas anti-fiber and anti-penton base antisera were negative. It is suggested that adenovirus may be neutralized by two pathways: aggregation of the virions (extracellular neutralization) as performed by anti-fiber antibodies and blocking of virion entrance from the acidic endosomes into the cytoplasm (intracellular neutralization). The latter effect could be obtained by (i) covering of the penton bases, as performed by anti-penton base antibodies, thereby preventing interaction between the penton bases and the endosomal membrane, which results in trapping of virions within endosomes, and (ii) inhibition of the low-pH-induced conformational change of the viral capsid, which seems to occur in the endosomes and is necessary for proper exposure of the penton bases, as performed by anti-hexon antibodies. Images PMID:3373570

  16. Adenovirus-based genetic vaccines for biodefense.

    PubMed

    Boyer, Julie L; Kobinger, Gary; Wilson, James M; Crystal, Ronald G

    2005-02-01

    The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

  17. Induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting anti-human adenovirus immunity.

    PubMed

    Hashimoto, Masahiko; Boyer, Julie L; Hackett, Neil R; Wilson, James M; Crystal, Ronald G

    2005-10-01

    Prevention or therapy for bioterrorism-associated anthrax infections requires rapidly acting effective vaccines. We recently demonstrated (Y. Tan, N. R. Hackett, J. L. Boyer, and R. G. Crystal, Hum. Gene Ther. 14:1673-1682, 2003) that a single administration of a recombinant serotype 5 adenovirus (Ad) vector expressing anthrax protective antigen (PA) provides rapid protection against anthrax lethal toxin challenge. However, approximately 35 to 50% of humans have preexisting neutralizing antibodies against Ad5. This study assesses the hypothesis that a recombinant adenovirus vaccine based on the nonhuman primate-derived serotype AdC7, against which humans do not have immunity, expressing PA (AdC7PA) will protect against anthrax lethal toxin even in the presence of preexisting anti-Ad5 immunity. Naive and Ad5-immunized BALB/c mice received (intramuscularly) 10(8) to 10(11) particle units (PU) of AdC7PA, Ad5PA (a human serotype Ad5-based vector expressing a secreted form of PA), or AdNull (an Ad5 vector with no transgene). Robust anti-PA immunoglobulin G and neutralizing antibodies were detected by 2 to 4 weeks following administration of AdC7PA to naive or Ad5 preimmunized mice, whereas low anti-PA titers were detected in Ad5-preimmunized mice following administration of Ad5PA. To assess protection in vivo, naive or mice previously immunized against Ad5 were immunized with AdC7PA or Ad5PA and then challenged with a lethal intravenous dose of Bacillus anthracis lethal toxin. Whereas Ad5PA protected naive mice against challenge with B. anthracis lethal toxin, Ad5PA was ineffective in mice that were previously immunized against Ad5. In contrast, AdC7PA functioned effectively not only to protect naive mice but also to protect Ad5-preimmunized mice, with 100% survival after lethal toxin challenge. These data suggest the nonhuman-based vector AdC7PA is an effective vaccine for the development of protective immunity against B. anthracis and importantly functions as a "sero

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

    PubMed Central

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

    2015-01-01

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

  19. Interferometric resolution boosting for spectrographs

    SciTech Connect

    Erskine, D J; Edelstein, J

    2004-05-25

    Externally dispersed interferometry (EDI) is a technique for enhancing the performance of spectrographs for wide bandwidth high resolution spectroscopy and Doppler radial velocimetry. By placing a small angle-independent interferometer near the slit of a spectrograph, periodic fiducials are embedded on the recorded spectrum. The multiplication of the stellar spectrum times the sinusoidal fiducial net creates a moir{acute e} pattern, which manifests high detailed spectral information heterodyned down to detectably low spatial frequencies. The latter can more accurately survive the blurring, distortions and CCD Nyquist limitations of the spectrograph. Hence lower resolution spectrographs can be used to perform high resolution spectroscopy and radial velocimetry. Previous demonstrations of {approx}2.5x resolution boost used an interferometer having a single fixed delay. We report new data indicating {approx}6x Gaussian resolution boost (140,000 from a spectrograph with 25,000 native resolving power), taken by using multiple exposures at widely different interferometer delays.

  20. Boosting Shift-Invariant Features

    NASA Astrophysics Data System (ADS)

    Hörnlein, Thomas; Jähne, Bernd

    This work presents a novel method for training shift-invariant features using a Boosting framework. Features performing local convolutions followed by subsampling are used to achieve shift-invariance. Other systems using this type of features, e.g. Convolutional Neural Networks, use complex feed-forward networks with multiple layers. In contrast, the proposed system adds features one at a time using smoothing spline base classifiers. Feature training optimizes base classifier costs. Boosting sample-reweighting ensures features to be both descriptive and independent. Our system has a lower number of design parameters as comparable systems, so adapting the system to new problems is simple. Also, the stage-wise training makes it very scalable. Experimental results show the competitiveness of our approach.

  1. 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.

  2. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

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

    2015-01-01

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

  3. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

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

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

  4. 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.

  5. A species of human alpha interferon that lacks the ability to boost human natural killer activity.

    PubMed Central

    Ortaldo, J R; Herberman, R B; Harvey, C; Osheroff, P; Pan, Y C; Kelder, B; Pestka, S

    1984-01-01

    Most species of recombinant leukocyte interferons (IFN-alpha A, -alpha B, -alpha C, -alpha D, -alpha F, -alpha I, and -alpha K) were capable of boosting human natural killer (NK) activity after a 2-hr treatment of cells at a concentration of 1-80 units/ml. In contrast, recombinant human IFN-alpha J was found to be incapable of augmenting NK activity after exposure of cells for 2 hr to concentrations as high as 10,000 units/ml. This inability of IFN-alpha J to boost NK activity was not complete because, after exposure of cells to a high concentration of IFN-alpha J (10,000 units/ml) for 18 hr, boosting of cytolysis was observed. IFN-alpha J appeared to interact with receptors for IFN on NK cells since it was found to interfere with the boosting of NK activity by other species of IFN-alpha. In contrast to its deficient ability to augment NK activity, IFN-alpha J has potent antiviral and antiproliferative activities. Such extensive dissociation of these biological activities has not been observed previously with any other natural or recombinant IFN species. Thus, this IFN species may be useful for evaluating the relative importance of various biological activities on the therapeutic effects of IFN, for understanding structure-function relationships, and for determining the biochemical pathways related to the various biological effects of IFN. PMID:6589637

  6. 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

  7. Online boosting for vehicle detection.

    PubMed

    Chang, Wen-Chung; Cho, Chih-Wei

    2010-06-01

    This paper presents a real-time vision-based vehicle detection system employing an online boosting algorithm. It is an online AdaBoost approach for a cascade of strong classifiers instead of a single strong classifier. Most existing cascades of classifiers must be trained offline and cannot effectively be updated when online tuning is required. The idea is to develop a cascade of strong classifiers for vehicle detection that is capable of being online trained in response to changing traffic environments. To make the online algorithm tractable, the proposed system must efficiently tune parameters based on incoming images and up-to-date performance of each weak classifier. The proposed online boosting method can improve system adaptability and accuracy to deal with novel types of vehicles and unfamiliar environments, whereas existing offline methods rely much more on extensive training processes to reach comparable results and cannot further be updated online. Our approach has been successfully validated in real traffic environments by performing experiments with an onboard charge-coupled-device camera in a roadway vehicle.

  8. 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.

  9. 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

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

    PubMed Central

    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

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

    PubMed

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

    1998-03-01

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

  12. 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

  13. Positive and negative regulation of adenovirus infection by CAR-like soluble protein, CLSP.

    PubMed

    Kawabata, K; Tashiro, K; Sakurai, F; Osada, N; Kusuda, J; Hayakawa, T; Yamanishi, K; Mizuguchi, H

    2007-08-01

    Coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin (Ig) superfamily and a component of epithelial tight junction. CAR also functions as a primary receptor for coxsackievirus B and adenovirus (Ad) infection. In this study, we report the identification of a novel protein, CAR-like soluble protein (CLSP), which is closely related to CAR. Mouse CLSP (mCLSP) was composed of 390 amino acids, including three Ig domains, and showed strong homology to the IgV domain of CAR. Interestingly, mCLSP lacks a transmembrane domain, indicating that this is a soluble protein. mCLSP mRNA was detected primarily in the brain and ovary. When mCLSP cDNA was introduced into SK HEP-1 cells, which were known to be CAR positive and easily infected with Ad vector, the infection with Ad vector was severely inhibited. On the other hand, mCLSP promoted the infection with Ad vector in CAR-negative NIH3T3 cells. Furthermore, recombinant CLSP directly bound to Ad and inhibited the Ad vector-mediated transduction in SK HEP-1 cells. Computational analysis for a genome database showed that the CLSP gene is rodent-specific, and that human and bovine lack this gene. These results suggest that CLSP may play a role in the antiviral defense of the host in rodent animals.

  14. Immunogenicity of seven new recombinant yellow fever viruses 17D expressing fragments of SIVmac239 Gag, Nef, and Vif in Indian rhesus macaques.

    PubMed

    Martins, Mauricio A; Bonaldo, Myrna C; Rudersdorf, Richard A; Piaskowski, Shari M; Rakasz, Eva G; Weisgrau, Kim L; Furlott, Jessica R; Eernisse, Christopher M; Veloso de Santana, Marlon G; Hidalgo, Bertha; Friedrich, Thomas C; Chiuchiolo, Maria J; Parks, Christopher L; Wilson, Nancy A; Allison, David B; Galler, Ricardo; Watkins, David I

    2013-01-01

    An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8(+) T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4(+) cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D - a clinically relevant vaccine vector - can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine

  15. 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

  16. 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)

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. Directed evolution of mutator adenoviruses resistant to antibody neutralization.

    PubMed

    Myers, Nicolle D; Skorohodova, Ksenia V; Gounder, Anshu P; Smith, Jason G

    2013-05-01

    We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

  20. 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

  1. 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

  2. Adenovirus Infections in Immunocompetent and Immunocompromised Patients

    PubMed Central

    2014-01-01

    SUMMARY Human adenoviruses (HAdVs) are an important cause of infections in both immunocompetent and immunocompromised individuals, and they continue to provide clinical challenges pertaining to diagnostics and treatment. The growing number of HAdV types identified by genomic analysis, as well as the improved understanding of the sites of viral persistence and reactivation, requires continuous adaptions of diagnostic approaches to facilitate timely detection and monitoring of HAdV infections. In view of the clinical relevance of life-threatening HAdV diseases in the immunocompromised setting, there is an urgent need for highly effective treatment modalities lacking major side effects. The present review summarizes the recent progress in the understanding and management of HAdV infections. PMID:24982316

  3. 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

  4. Electric rockets get a boost

    SciTech Connect

    Ashley, S.

    1995-12-01

    This article reports that xenon-ion thrusters are expected to replace conventional chemical rockets in many nonlaunch propulsion tasks, such as controlling satellite orbits and sending space probes on long exploratory missions. The space age dawned some four decades ago with the arrival of powerful chemical rockets that could propel vehicles fast enough to escape the grasp of earth`s gravity. Today, chemical rocket engines still provide the only means to boost payloads into orbit and beyond. The less glamorous but equally important job of moving vessels around in space, however, may soon be assumed by a fundamentally different rocket engine technology that has been long in development--electric propulsion.

  5. Recursive bias estimation and L2 boosting

    SciTech Connect

    Hengartner, Nicolas W; Cornillon, Pierre - Andre; Matzner - Lober, Eric

    2009-01-01

    This paper presents a general iterative bias correction procedure for regression smoothers. This bias reduction schema is shown to correspond operationally to the L{sub 2} Boosting algorithm and provides a new statistical interpretation for L{sub 2} Boosting. We analyze the behavior of the Boosting algorithm applied to common smoothers S which we show depend on the spectrum of I - S. We present examples of common smoother for which Boosting generates a divergent sequence. The statistical interpretation suggest combining algorithm with an appropriate stopping rule for the iterative procedure. Finally we illustrate the practical finite sample performances of the iterative smoother via a simulation study.

  6. Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector.

    PubMed

    Pratt, William D; Wang, Danher; Nichols, Donald K; Luo, Min; Woraratanadharm, Jan; Dye, John M; Holman, David H; Dong, John Y

    2010-04-01

    Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

  7. ADENO-ASSOCIATED SATELLITE VIRUS INTERFERENCE WITH THE REPLICATION OF ITS HELPER ADENOVIRUS

    PubMed Central

    Parks, Wade P.; Casazza, Anna M.; Alcott, Judith; Melnick, Joseph L.

    1968-01-01

    Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80°C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This

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

    PubMed

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

    2007-06-01

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

  9. 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

  10. 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.

  11. Complete genome analysis of a novel E3-partial-deleted human adenovirus type 7 strain isolated in Southern China.

    PubMed

    Su, Xiaobo; Tian, Xingui; Zhang, Qiwei; Li, Haitao; Li, Xiao; Sheng, Huiying; Wang, Youshao; Wu, Houbo; Zhou, Rong

    2011-01-01

    Human adenovirus (HAdV) is a causative agent of acute respiratory disease, which is prevalent throughout the world. Recently there are some reports which found that the HAdV-3 and HAdV-5 genomes were very stable across 50 years of time and space. But more and more recombinant genomes have been identified in emergent HAdV pathogens and it is a pathway for the molecular evolution of types. In our paper, we found a HAdV-7 GZ07 strain isolated from a child with acute respiratory disease, whose genome was E3-partial deleted. The whole genome was 32442 bp with 2864 bp deleted in E3 region and was annotated in detail (GenBank: HQ659699). The growth character was the same as that of another HAdV-7 wild strain which had no gene deletion. By comparison with E3 regions of the other HAdV-B, we found that only left-end two proteins were remained: 12.1 kDa glycoprotein and 16.1 kDa protein. E3 MHC class I antigen-binding glycoprotein, hypothetical 20.6 kDa protein, 20.6 kDa protein, 7.7 kDa protein., 10.3 kDa protein, 14.9 kDa protein and E3 14.7 kDa protein were all missing. It is the first report about E3 deletion in human adenovirus, which suggests that E3 region is also a possible recombination region in adenovirus molecular evolution.

  12. High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system.

    PubMed Central

    Alkhatib, G; Briedis, D J

    1988-01-01

    The entire measles virus (MV) hemagglutinin (HA)-coding region was reconstructed from cloned cDNAs and used as part of a hybrid transcription unit to replace a region of the adenovirus type 5 genome corresponding to the entire E1a transcription unit and most of the E1b transcription unit. The resulting recombinant virus was stable and able to replicate to high titers in 293 cells (which constitutively express the complementary E1a-E1b functions) in the absence of helper virus. During infection of 293 cells, the hybrid virus expressed MV HA protein which was indistinguishable from that expressed in MV-infected cells in terms of immunoreactivity, gel mobility, glycosylation, subcellular localization, and biologic activity. Infection of 293 cells with the hybrid virus led to high-level synthesis of the MV HA protein (equivalent to 65 to 130% of the level seen in MV-infected cells). At late times after high-multiplicity hybrid virus infection of HeLa and Vero cells (which do not express E1 functions), the level of HA protein synthesis was at least 35% of that seen in 293 cells. This MV-adenovirus recombinant will be useful in the study of the biologic properties of the MV HA protein and in assessment of the potential usefulness of hybrid adenoviruses as live-virus vaccine vectors. Images PMID:3292790

  13. Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

    PubMed Central

    Teixeira, Lais H.; Tararam, Cibele A.; Lasaro, Marcio O.; Camacho, Ariane G. A.; Ersching, Jonatan; Leal, Monica T.; Herrera, Sócrates; Bruna-Romero, Oscar; Soares, Irene S.; Nussenzweig, Ruth S.; Ertl, Hildegund C. J.; Nussenzweig, Victor

    2014-01-01

    Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine. PMID:24478093

  14. Series Connected Buck-Boost Regulator

    NASA Technical Reports Server (NTRS)

    Birchenough, Arthur G. (Inventor)

    2006-01-01

    A Series Connected Buck-Boost Regulator (SCBBR) that switches only a fraction of the input power, resulting in relatively high efficiencies. The SCBBR has multiple operating modes including a buck, a boost, and a current limiting mode, so that an output voltage of the SCBBR ranges from below the source voltage to above the source voltage.

  15. Bagging, boosting, and C4.5

    SciTech Connect

    Quinlan, J.R.

    1996-12-31

    Breiman`s bagging and Freund and Schapire`s boosting are recent methods for improving the predictive power of classifier learning systems. Both form a set of classifiers that are combined by voting, bagging by generating replicated bootstrap samples of the data, and boosting by adjusting the weights of training instances. This paper reports results of applying both techniques to a system that learns decision trees and testing on a representative collection of datasets. While both approaches substantially improve predictive accuracy, boosting shows the greater benefit. On the other hand, boosting also produces severe degradation on some datasets. A small change to the way that boosting combines the votes of learned classifiers reduces this downside and also leads to slightly better results on most of the datasets considered.

  16. Boosting human learning by hypnosis.

    PubMed

    Nemeth, Dezso; Janacsek, Karolina; Polner, Bertalan; Kovacs, Zoltan Ambrus

    2013-04-01

    Human learning and memory depend on multiple cognitive systems related to dissociable brain structures. These systems interact not only in cooperative but also sometimes competitive ways in optimizing performance. Previous studies showed that manipulations reducing the engagement of frontal lobe-mediated explicit attentional processes could lead to improved performance in striatum-related procedural learning. In our study, hypnosis was used as a tool to reduce the competition between these 2 systems. We compared learning in hypnosis and in the alert state and found that hypnosis boosted striatum-dependent sequence learning. Since frontal lobe-dependent processes are primarily affected by hypnosis, this finding could be attributed to the disruption of the explicit attentional processes. Our result sheds light not only on the competitive nature of brain systems in cognitive processes but also could have important implications for training and rehabilitation programs, especially for developing new methods to improve human learning and memory performance.

  17. Advanced Airfoils Boost Helicopter Performance

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Carson Helicopters Inc. licensed the Langley RC4 series of airfoils in 1993 to develop a replacement main rotor blade for their Sikorsky S-61 helicopters. The company's fleet of S-61 helicopters has been rebuilt to include Langley's patented airfoil design, and the helicopters are now able to carry heavier loads and fly faster and farther, and the main rotor blades have twice the previous service life. In aerial firefighting, the performance-boosting airfoils have helped the U.S. Department of Agriculture's Forest Service control the spread of wildfires. In 2003, Carson Helicopters signed a contract with Ducommun AeroStructures Inc., to manufacture the composite blades for Carson Helicopters to sell

  18. Boost-phase discrimination research

    NASA Technical Reports Server (NTRS)

    Langhoff, Stephen R.; Feiereisen, William J.

    1993-01-01

    The final report describes the combined work of the Computational Chemistry and Aerothermodynamics branches within the Thermosciences Division at NASA Ames Research Center directed at understanding the signatures of shock-heated air. Considerable progress was made in determining accurate transition probabilities for the important band systems of NO that account for much of the emission in the ultraviolet region. Research carried out under this project showed that in order to reproduce the observed radiation from the bow shock region of missiles in their boost phase it is necessary to include the Burnett terms in the constituent equation, account for the non-Boltzmann energy distribution, correctly model the NO formation and rotational excitation process, and use accurate transition probabilities for the NO band systems. This work resulted in significant improvements in the computer code NEQAIR that models both the radiation and fluid dynamics in the shock region.

  19. Heterologous Prime-Boost HIV-1 Vaccination Regimens in Pre-Clinical and Clinical Trials

    PubMed Central

    Brown, Scott A.; Surman, Sherri L.; Sealy, Robert; Jones, Bart G.; Slobod, Karen S.; Branum, Kristen; Lockey, Timothy D.; Howlett, Nanna; Freiden, Pamela; Flynn, Patricia; Hurwitz, Julia L.

    2010-01-01

    Currently, there are more than 30 million people infected with HIV-1 and thousands more are infected each day. Vaccination is the single most effective mechanism for prevention of viral disease, and after more than 25 years of research, one vaccine has shown somewhat encouraging results in an advanced clinical efficacy trial. A modified intent-to-treat analysis of trial results showed that infection was approximately 30% lower in the vaccine group compared to the placebo group. The vaccine was administered using a heterologous prime-boost regimen in which both target antigens and delivery vehicles were changed during the course of inoculations. Here we examine the complexity of heterologous prime-boost immunizations. We show that the use of different delivery vehicles in prime and boost inoculations can help to avert the inhibitory effects caused by vector-specific immune responses. We also show that the introduction of new antigens into boost inoculations can be advantageous, demonstrating that the effect of ‘original antigenic sin’ is not absolute. Pre-clinical and clinical studies are reviewed, including our own work with a three-vector vaccination regimen using recombinant DNA, virus (Sendai virus or vaccinia virus) and protein. Promising preliminary results suggest that the heterologous prime-boost strategy may possibly provide a foundation for the future prevention of HIV-1 infections in humans. PMID:20407589

  20. Adenovirus-associated deaths in US military during postvaccination period, 1999-2010.

    PubMed

    Potter, Robert N; Cantrell, Joyce A; Mallak, Craig T; Gaydos, Joel C

    2012-03-01

    Adenoviruses are frequent causes of respiratory disease in the US military population. A successful immunization program against adenovirus types 4 and 7 was terminated in 1999. Review of records in the Mortality Surveillance Division, Armed Forces Medical Examiner System, identified 8 deaths attributed to adenovirus infections in service members during 1999-2010.

  1. Characterization of a new adenovirus isolated from black-tailed deer in California.

    PubMed

    Lehmkuhl, H D; Hobbs, L A; Woods, L W

    2001-01-01

    An adenovirus associated with systemic and localized vascular damage was demonstrated by transmission electron microscopy and immunohistochemistry in a newly recognized epizootic hemorrhagic disease in California black-tailed deer. In this study, we describe the cultural, physicochemical and serological characteristics of a virus isolated from lung using neonatal white-tail deer lung and turbinate cell cultures. The virus had the cultural, morphological and physicochemical characteristics of members of the Adenoviridae family. The virus would not replicate in low passage fetal bovine, caprine or ovine cells. Antiserum to the deer adenovirus, strain D94-2569, neutralized bovine adenovirus type-6 (BAdV-6), BAdV-7, and caprine adenovirus type-1 (GAdV-1). Antiserum to BAdV-6 did not neutralize the deer adenovirus but antiserum to BAdV-7 and GAdV-1 neutralized the deer adenovirus. Cross-neutralization with the other bovine, caprine and ovine adenovirus species was not observed. Restriction endonuclease patterns generated for the deer adenovirus were unique compared to those for the currently recognized bovine, caprine and ovine adenovirus types. Amino acid sequence alignments of the hexon gene from the deer adenovirus strain D94-2569 indicate that it is a member of the proposed new genus (Atadenovirus) of the Adenoviridae family. While closely related antigenically to BAdV-7 and GAdV-1, the deer adenovirus appears sufficiently distinct culturally and molecularly to justify consideration as a new adenovirus type.

  2. 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

  3. Enhanced UV inactivation of adenoviruses under polychromatic UV lamps.

    PubMed

    Linden, Karl G; Thurston, Jeanette; Schaefer, Raymond; Malley, James P

    2007-12-01

    Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. 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 published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm(-2) and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm(-2). The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed. PMID:17933932

  4. 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.

  5. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  6. [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

  7. Adenovirus Pneumonia Complicated With Acute Respiratory Distress Syndrome

    PubMed Central

    Hung, Ka-Ho; Lin, Lung-Huang

    2015-01-01

    Abstract Severe adenovirus infection in children can manifest with acute respiratory distress syndrome (ARDS) and respiratory failure, leading to the need for prolonged mechanical support in the form of either mechanical ventilation or extracorporeal life support. Early extracorporeal membrane oxygenation (ECMO) intervention for children with ARDS should be considered if selection criteria fulfill. We report on a 9-month-old boy who had adenovirus pneumonia with rapid progression to ARDS. Real-time polymerase chain reaction tests of sputum and pleural effusion samples confirmed adenovirus serotype 7. Chest x-rays showed progressively increasing infiltrations and pleural effusions in both lung fields within 11 days. Because conventional ARDS therapies failed, we initiated ECMO with high-frequency oscillatory ventilation (HFOV) for 9 days. Chest x-rays showed gradual improvements in lung expansion. This patient was subsequently discharged after a hospital stay of 38 days. Post-ECMO and adenovirus sequelae were followed in our outpatient department. Adenovirus pneumonia in children can manifest with severe pulmonary morbidity and respiratory failure. The unique lung recruitment by HFOV can be a useful therapeutic option for severe ARDS patients when combined with sufficient lung rest provided by ECMO. PMID:25997046

  8. New Insights on Adenovirus as Vaccine Vectors

    PubMed Central

    Lasaro, Marcio O; Ertl, Hildegund CJ

    2009-01-01

    Adenovirus (Ad) vectors were initially developed for treatment of genetic diseases. Their usefulness for permanent gene replacement was limited by their high immunogenicity, which resulted in rapid elimination of transduced cells through induction of T and B cells to antigens of Ad and the transgene product. The very trait that excluded their use for sustained treatment of genetic diseases made them highly attractive as vaccine carriers. Recently though results showed that Ad vectors based on common human serotypes, such as serotype 5, may not be ideal as vaccine carriers. A recently conducted phase 2b trial, termed STEP trial, with an AdHu5-based vaccine expressing antigens of human immunodeficiency virus 1 (HIV-1) not only showed lack of efficacy in spite of the vaccine's immunogenicity, but also suggested an increased trend for HIV acquisition in individuals that had circulating AdHu5 neutralizing antibodies prior to vaccination. Alternative serotypes from humans or nonhuman primates (NHPs), to which most humans lack pre-existing immunity, have been vectored and may circumvent the problems encountered with the use of AdHu5 vectors in humans. In summary, although Ad vectors have seen their share of setbacks in recent years, they remain viable tools for prevention or treatment of a multitude of diseases. PMID:19513019

  9. Viability of human adenovirus from hospital fomites.

    PubMed

    Ganime, Ana Carolina; Carvalho-Costa, Filipe A; Santos, Marisa; Costa Filho, Rubens; Leite, José Paulo G; Miagostovich, Marize P

    2014-12-01

    The monitoring of environmental microbial contamination in healthcare facilities may be a valuable tool to determine pathogens transmission in those settings; however, such procedure is limited to bacterial indicators. Viruses are found commonly in those environments and are rarely used for these procedures. The aim of this study was to assess distribution and viability of a human DNA virus on fomites in an Adult Intensive Care Unit of a private hospital in Rio de Janeiro, Brazil. Human adenoviruses (HAdV) were investigated in 141 fomites by scraping the surface area and screening by quantitative PCR (qPCR) using TaqMan® System (Carlsbad, CA). Ten positive samples were selected for virus isolation in A549 and/or HEp2c cell lines. A total of 63 samples (44.7%) were positive and presented viral load ranging from 2.48 × 10(1) to 2.1 × 10(3) genomic copies per millilitre (gc/ml). The viability was demonstrated by integrated cell culture/nested-PCR in 5 out of 10 samples. Nucleotide sequencing confirmed all samples as HAdV and characterized one of them as specie B, serotype 3 (HAdV-3). The results indicate the risk of nosocomial transmission via contaminated fomites and point out the use of HAdV as biomarkers of environmental contamination.

  10. 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

  11. 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

  12. Selection of nonfastidious adenovirus species in 293 cells inoculated with stool specimens containing adenovirus 40.

    PubMed

    Brown, M

    1985-08-01

    Of 35 stool specimens isolated and examined in 293 cells, 15 isolates contained adenovirus species 40 (Ad40), and 4 of these 15 isolates also contained a nonfastidious adenovirus species (Ad1 in two cases, Ad18 or Ad31) which was selected over Ad40 during serial passage in the 293 cells. The selection of Ad1 over Ad40 was examined in detail. Restriction analysis of intracellular DNA and the relative infectivity titers of Ad40 and Ad1 at each passage level after the inoculation of 293 cells with a particular stool specimen demonstrated that although the amount of Ad40 DNA synthesized far exceeded that of Ad1, the relative infectivity titer of Ad40 was low. The growth characteristics of Ad40 were then compared with those of Ad1, Ad18, and Ad41 in singly infected 293 cell cultures. One-step growth curves showed the same growth rate in each case, with a latent period of 12 h and a maximum titer at 24 to 36 h postinfection. Yields of infectious Ad40 virus were consistently 100- to 1,000-fold lower than those of Ad1. This difference was reflected by a reduced yield of total AD40 virions (p1.34) as determined by 35S labeling experiments. However, the 3- to 10-fold reduction in total yield of Ad40 virions did not account for the 100- to 1,000-fold reduction in the yield of infectious virus. PMID:2993350

  13. 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

  14. Riemann curvature of a boosted spacetime geometry

    NASA Astrophysics Data System (ADS)

    Battista, Emmanuele; Esposito, Giampiero; Scudellaro, Paolo; Tramontano, Francesco

    2016-10-01

    The ultrarelativistic boosting procedure had been applied in the literature to map the metric of Schwarzschild-de Sitter spacetime into a metric describing de Sitter spacetime plus a shock-wave singularity located on a null hypersurface. This paper evaluates the Riemann curvature tensor of the boosted Schwarzschild-de Sitter metric by means of numerical calculations, which make it possible to reach the ultrarelativistic regime gradually by letting the boost velocity approach the speed of light. Thus, for the first time in the literature, the singular limit of curvature, through Dirac’s δ distribution and its derivatives, is numerically evaluated for this class of spacetimes. Moreover, the analysis of the Kretschmann invariant and the geodesic equation shows that the spacetime possesses a “scalar curvature singularity” within a 3-sphere and it is possible to define what we here call “boosted horizon”, a sort of elastic wall where all particles are surprisingly pushed away, as numerical analysis demonstrates. This seems to suggest that such “boosted geometries” are ruled by a sort of “antigravity effect” since all geodesics seem to refuse to enter the “boosted horizon” and are “reflected” by it, even though their initial conditions are aimed at driving the particles toward the “boosted horizon” itself. Eventually, the equivalence with the coordinate shift method is invoked in order to demonstrate that all δ2 terms appearing in the Riemann curvature tensor give vanishing contribution in distributional sense.

  15. Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extend the number of species within the genus Aviadenovirus.

    PubMed

    Marek, Ana; Kaján, Győző L; Kosiol, Carolin; Harrach, Balázs; Schlötterer, Christian; Hess, Michael

    2014-08-01

    Complete genomes of the first isolates of pigeon adenovirus 1 (PiAdV-1) and Muscovy duck adenovirus (duck adenovirus 2, DAdV-2) were sequenced. The PiAdV-1 genome is 45,480bp long, and has a gene organization most similar to turkey adenovirus 1. Near the left end of the genome, it lacks ORF0, ORF1A, ORF1B and ORF1C, and possesses ORF52, whereas six novel genes were found near the right end. The DAdV-2 genome is 43,734bp long, and has a gene organization similar to that of goose adenovirus 4 (GoAdV-4). It lacks ORF51, ORF1C and ORF54, and possesses ORF55A and five other novel genes. PiAdV-1 and DAdV-2 genomes contain two and one fiber genes, respectively. Genome organization, G+C content, molecular phylogeny and host type confirm the need to establish two novel species (Pigeon aviadenovirus A and Duck aviadenovirus B) within the genus Aviadenovirus. Phylogenetic data show that DAdV-2 is most closely related to GoAdV-4.

  16. 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

  17. 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

  18. 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

  19. 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

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

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

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

  1. 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.

  2. Boosting domain wall propagation by notches

    NASA Astrophysics Data System (ADS)

    Yuan, H. Y.; Wang, X. R.

    2015-08-01

    We report a counterintuitive finding that notches in an otherwise homogeneous magnetic nanowire can boost current-induced domain wall (DW) propagation. DW motion in notch-modulated wires can be classified into three phases: (1) A DW is pinned around a notch when the current density is below the depinning current density. (2) DW propagation velocity is boosted by notches above the depinning current density and when nonadiabatic spin-transfer torque strength β is smaller than the Gilbert damping constant α . The boost can be multifold. (3) DW propagation velocity is hindered when β >α . The results are explained by using the Thiele equation.

  3. 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. PMID:27635215

  4. 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

  5. Effects of cold atmospheric plasmas on adenoviruses in solution

    NASA Astrophysics Data System (ADS)

    Zimmermann, J. L.; Dumler, K.; Shimizu, T.; Morfill, G. E.; Wolf, A.; Boxhammer, V.; Schlegel, J.; Gansbacher, B.; Anton, M.

    2011-12-01

    Experiments were performed with cold atmospheric plasma (CAP) to inactivate adenovirus, a non-enveloped double stranded DNA virus, in solution. The plasma source used was a surface micro-discharge technology operating in air. Various plasma diagnostic measurements and tests were performed in order to determine the efficacy of CAPs and to understand the inactivation mechanism(s). Different stages of the adenovirus ‘life cycle’ were investigated—infectivity and gene expression as well as viral replication and spread. Within 240 s of CAP treatment, inactivation of up to 6 decimal log levels can be achieved.

  6. Generation of E3-deleted canine adenovirus type 2 expressing the Gc glycoprotein of Seoul virus by gene insertion or deletion of related terminal region sequences.

    PubMed

    Yuan, Zi-Guo; Luo, Sheng-Jun; Xu, Hui-Juan; Wang, Xiao-Hu; Li, Juan; Yuan, Li-Guo; He, Le-Tian; Zhang, Xiu-Xiang

    2010-07-01

    Seoul virus (SEOV) is one of the four hantaviruses known to cause haemorrhagic fever with renal syndrome. The medium genome segment encodes the Gn/Gc glycoproteins of SEOV, which form the major structural part of the virus envelope. Gc and/or Gn are the candidate antigens of hantavirus for induction of a highly immunogenic response for hantavirus vaccine. In this study, the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gc protein of SEOV was evaluated in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against an intensive infectious challenge with approximately 1000 50 % infective doses for SEOV strain CC-2. Protective-level neutralizing antibodies were maintained for at least 20 weeks. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

  7. 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

  8. Bovine adenovirus type 10 identified in fatal cases of adenovirus-associated enteric disease in cattle by in situ hybridization.

    PubMed Central

    Smyth, J A; Benkö, M; Moffett, D A; Harrach, B

    1996-01-01

    A severe, naturally occurring enteric disease of cattle in which adenovirus inclusions are present in the intestinal vascular endothelium has been recognized in several countries; three different adenovirus serotypes have been isolated from affected animals. An in situ hybridization technique for the detection of bovine adenoviral DNA was developed and applied to tissue from 13 cattle in Northern Ireland diagnosed to have the disease. Bovine adenovirus serotype 10 (BAV-10) was identified in the vascular inclusions of all cattle, providing strong evidence that adenoviral enteric vascular disease in cattle is associated with this serotype. The existence of BAV-10 has only recently been recognized. The first molecular biology-based technique for the diagnosis of BAV-10 infection is described. The animals in the present study are the first in which BAV-10 has had a confirmed role in a pathologic process. PMID:8727916

  9. CD46 Is a Cellular Receptor for All Species B Adenoviruses except Types 3 and 7

    PubMed Central

    Marttila, Marko; Persson, David; Gustafsson, Dan; Liszewski, M. Kathryn; Atkinson, John P.; Wadell, Göran; Arnberg, Niklas

    2005-01-01

    The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7. PMID:16254377

  10. 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.

  11. 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

  12. Adenovirus Respiratory Tract Infections in Peru

    PubMed Central

    Ampuero, Julia S.; Ocaña, Víctor; Gómez, Jorge; Gamero, María E.; Garcia, Josefina; Halsey, Eric S.; Laguna-Torres, V. Alberto

    2012-01-01

    Background Currently, there is a paucity of data regarding human adenovirus (HAdv) circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. Methods/Principal Findings Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI) or severe acute respiratory infection (SARI) were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. Conclusions/Significance HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness. PMID:23056519

  13. Adenovirus Dodecahedron, as a Drug Delivery Vector

    PubMed Central

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

    2009-01-01

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

  14. Skip Dinner and Maybe Boost Your Metabolism

    MedlinePlus

    ... 161845.html Skip Dinner and Maybe Boost Your Metabolism But, study didn't show overall changes in ... has an internal clock, and many aspects of metabolism are working best in the morning, according to ...

  15. Old Drug Boosts Brain's Memory Centers

    MedlinePlus

    ... gov/news/fullstory_159605.html Old Drug Boosts Brain's Memory Centers But more research needed before recommending ... called methylene blue may rev up activity in brain regions involved in short-term memory and attention, ...

  16. Avoiding Anemia: Boost Your Red Blood Cells

    MedlinePlus

    ... link, please review our exit disclaimer . Subscribe Avoiding Anemia Boost Your Red Blood Cells If you’re ... and sluggish, you might have a condition called anemia. Anemia is a common blood disorder that many ...

  17. Tools to Boost Steam System Efficiency

    SciTech Connect

    2005-05-01

    The Steam System Scoping Tool quickly evaluates your entire steam system operation and spots the areas that are the best opportunities for improvement. The tool suggests a range of ways to save steam energy and boost productivity.

  18. Relativistic projection and boost of solitons

    SciTech Connect

    Wilets, L.

    1991-01-01

    This report discusses the following topics on the relativistic projection and boost of solitons: The center of mass problem; momentum eigenstates; variation after projection; and the nucleon as a composite. (LSP).

  19. Relativistic projection and boost of solitons

    SciTech Connect

    Wilets, L.

    1991-12-31

    This report discusses the following topics on the relativistic projection and boost of solitons: The center of mass problem; momentum eigenstates; variation after projection; and the nucleon as a composite. (LSP).

  20. Anemia Boosts Stroke Death Risk, Study Finds

    MedlinePlus

    ... page: https://medlineplus.gov/news/fullstory_160476.html Anemia Boosts Stroke Death Risk, Study Finds Blood condition ... 2016 (HealthDay News) -- Older stroke victims suffering from anemia -- a lack of red blood cells -- may have ...

  1. 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.

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

    PubMed

    Bodewes, R; van de Bildt, M W G; Schapendonk, C M E; van Leeuwen, M; van Boheemen, S; de Jong, A A W; 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.

  3. 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

  4. 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

  5. Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35

    PubMed Central

    Tuve, Sebastian; Wang, Hongjie; Jacobs, Jeffrey D.; Yumul, Roma C.; Smith, David F.; Lieber, André

    2008-01-01

    Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection. PMID:18974862

  6. 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

  7. 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

  8. Centaur liquid oxygen boost pump vibration test

    NASA Technical Reports Server (NTRS)

    Tang, H. M.

    1975-01-01

    The Centaur LOX boost pump was subjected to both the simulated Titan Centaur proof flight and confidence demonstration vibration test levels. For each test level, both sinusoidal and random vibration tests were conducted along each of the three orthogonal axes of the pump and turbine assembly. In addition to these tests, low frequency longitudinal vibration tests for both levels were conducted. All tests were successfully completed without damage to the boost pump.

  9. Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

    PubMed

    Chen, Weizao; Liu, Mingqiu; Jiao, Ye; Yan, Weiyao; Wei, Xuefeng; Chen, Jiulian; Fei, Liang; Liu, Yang; Zuo, Xiaoping; Yang, Fugui; Lu, Yonggan; Zheng, Zhaoxin

    2006-04-01

    Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD. PMID:16537624

  10. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

    PubMed

    Griffin, Bryan D; Nagy, Eva

    2011-06-01

    Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

  11. 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.

  12. 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

  13. Age at Mycobacterium bovis BCG Priming Has Limited Impact on Anti-Tuberculosis Immunity Boosted by Respiratory Mucosal AdHu5Ag85A Immunization in a Murine Model.

    PubMed

    Damjanovic, Daniela; Khera, Amandeep; Afkhami, Sam; Lai, Rocky; Zganiacz, Anna; Jeyanathan, Mangalakumari; Xing, Zhou

    2015-01-01

    Tuberculosis (TB) remains a global pandemic despite the use of Bacillus Calmette-Guérin (BCG) vaccine, partly because BCG fails to effectively control adult pulmonary TB. The introduction of novel boost vaccines such as the human Adenovirus 5-vectored AdHu5Ag85A could improve and prolong the protective immunity of BCG immunization. Age at which BCG immunization is implemented varies greatly worldwide, and research is ongoing to discover the optimal stage during childhood to administer the vaccine, as well as when to boost the immune response with potential novel vaccines. Using a murine model of subcutaneous BCG immunization followed by intranasal AdHu5Ag85A boosting, we investigated the impact of age at BCG immunization on protective efficacy of BCG prime and AdHu5Ag85A boost immunization-mediated protection. Our results showed that age at parenteral BCG priming has limited impact on the efficacy of BCG prime-AdHu5Ag85A respiratory mucosal boost immunization-enhanced protection. However, when BCG immunization was delayed until the maturity of the immune system, longer sustained memory T cells were generated and resulted in enhanced boosting effect on T cells of AdHu5Ag85A respiratory mucosal immunization. Our findings hold implications for the design of new TB immunization protocols for humans.

  14. 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

  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.

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

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

    PubMed

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

    2015-06-01

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

  2. 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...

  3. Adenovirus infection reverses the antiviral state induced by human interferon.

    PubMed

    Feduchi, E; Carrasco, L

    1987-04-01

    HeLa cells treated with human lymphoblastoid interferon do not synthesize poliovirus proteins. The antiviral state against poliovirus is reversed if cells are previously infected with adenovirus type 5. A late gene product seems to be involved in this reversion, since no effect is observed at early stages of infection or in the presence of aphidicolin.

  4. 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

  5. Isolation of adenovirus from lambs with upper respiratory syndrome.

    PubMed

    Pommer, J; Schamber, G

    1991-07-01

    The role of viruses in the etiology of recurrent upper respiratory disease in newly weaned lambs was studied during 1984-1985 at the North Dakota Sheep Experiment Station. Serum samples collected from lambs at weaning, from lambs with signs of respiratory disease, and 3 weeks following the onset of clinical signs were tested for antibodies to ovine adenovirus (OAV), respiratory syncytial virus (RSV), and parainfluenza type-3 virus (PI-3). Virus isolation studies were performed on nasal secretions samples taken at the same time. Parainfluenza type-3 was isolated from 1 of 275 lambs tested, and there was 2.5% overall 4-fold increase in antibody titer to PI-3 during the 2-year study. An adenovirus with a different restriction endonuclease digestion pattern from that previously reported adenovirus strains in the United States was isolated from 13 of 275 nasal secretions collected from lambs at the time of weaning. There was a 17.6% overall 4-fold increase in seroconversion to the adenovirus isolated from the lambs with clinical disease.

  6. Tumorigenicity and adenovirus-transformed cells: Collagen interaction and cell surface laminin are controlled by the serotype origin of the E1A and E1B genes

    SciTech Connect

    Bober, F.J.; Birk, D.E.; Raska, K. Jr. ); Shenk, T. )

    1988-02-01

    A library of cells transformed with recombinant adenoviruses was used to study tumorigenicity and interaction with extracellular matrix. Cells expressing the complete E1 region of highly oncogenic adenovirus type 12 (Ad12) are tumorigenic, adhere preferentially to type IV collagen, and express cell surface laminin. Weakly tumorigenic cells, which express the E1A oncogene of Ad12 and the E1B genes of Ad5, also attach preferentially to type IV collagen but do not contain laminin on their surface. Cells which express the E1A oncogene of Ad5 and the E1B genes of Ad12 are nontumorigenic and do not preferentially attach to type IV versus type I collagen but have laminin on their surface. There is no significant difference in the amounts of laminin secreted into the culture medium among cells expressing the E1B genes of Ad5 or Ad12. In vitro assays show that cells which express the E1B genes of Ad12, irrespective of the origin of the E1A genes, can bind three times more exogenously added {sup 125}I-laminin than cells expressing the E1B genes of nononcogenic Ad5. The interaction of adenovirus-transformed cells with collagen is controlled by the serotype origin of the E1A oncogene, whereas cell surface laminin is controlled by the serotype origin of the E1B genes.

  7. A combinatory strategy for detection of live CTCs using microfiltration and a new telomerase-selective adenovirus

    PubMed Central

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

    2015-01-01

    Circulating tumor cells (CTCs) 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 cancer patients. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared to 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 (FMSAs) 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 breast and pancreatic cancer patients, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. PMID:25589497

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

    PubMed

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

    2005-12-20

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

  10. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    PubMed

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

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

    PubMed

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

    2005-12-20

    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings. PMID:16169033

  12. 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

  13. Tracking down hyper-boosted top quarks

    SciTech Connect

    Larkoski, Andrew J.; Maltoni, Fabio; Selvaggi, Michele

    2015-06-05

    The identification of hadronically decaying heavy states, such as vector bosons, the Higgs, or the top quark, produced with large transverse boosts has been and will continue to be a central focus of the jet physics program at the Large Hadron Collider (LHC). At a future hadron collider working at an order-of-magnitude larger energy than the LHC, these heavy states would be easily produced with transverse boosts of several TeV. At these energies, their decay products will be separated by angular scales comparable to individual calorimeter cells, making the current jet substructure identification techniques for hadronic decay modes not directly employable. In addition, at the high energy and luminosity projected at a future hadron collider, there will be numerous sources for contamination including initial- and final-state radiation, underlying event, or pile-up which must be mitigated. We propose a simple strategy to tag such "hyper-boosted" objects that defines jets with radii that scale inversely proportional to their transverse boost and combines the standard calorimetric information with charged track-based observables. By means of a fast detector simulation, we apply it to top quark identification and demonstrate that our method efficiently discriminates hadronically decaying top quarks from light QCD jets up to transverse boosts of 20 TeV. Lastly, our results open the way to tagging heavy objects with energies in the multi-TeV range at present and future hadron colliders.

  14. Tracking down hyper-boosted top quarks

    DOE PAGESBeta

    Larkoski, Andrew J.; Maltoni, Fabio; Selvaggi, Michele

    2015-06-05

    The identification of hadronically decaying heavy states, such as vector bosons, the Higgs, or the top quark, produced with large transverse boosts has been and will continue to be a central focus of the jet physics program at the Large Hadron Collider (LHC). At a future hadron collider working at an order-of-magnitude larger energy than the LHC, these heavy states would be easily produced with transverse boosts of several TeV. At these energies, their decay products will be separated by angular scales comparable to individual calorimeter cells, making the current jet substructure identification techniques for hadronic decay modes not directlymore » employable. In addition, at the high energy and luminosity projected at a future hadron collider, there will be numerous sources for contamination including initial- and final-state radiation, underlying event, or pile-up which must be mitigated. We propose a simple strategy to tag such "hyper-boosted" objects that defines jets with radii that scale inversely proportional to their transverse boost and combines the standard calorimetric information with charged track-based observables. By means of a fast detector simulation, we apply it to top quark identification and demonstrate that our method efficiently discriminates hadronically decaying top quarks from light QCD jets up to transverse boosts of 20 TeV. Lastly, our results open the way to tagging heavy objects with energies in the multi-TeV range at present and future hadron colliders.« less

  15. Tracking down hyper-boosted top quarks

    NASA Astrophysics Data System (ADS)

    Larkoski, Andrew J.; Maltoni, Fabio; Selvaggi, Michele

    2015-06-01

    The identification of hadronically decaying heavy states, such as vector bosons, the Higgs, or the top quark, produced with large transverse boosts has been and will continue to be a central focus of the jet physics program at the Large Hadron Collider (LHC). At a future hadron collider working at an order-of-magnitude larger energy than the LHC, these heavy states would be easily produced with transverse boosts of several TeV. At these energies, their decay products will be separated by angular scales comparable to individual calorimeter cells, making the current jet substructure identification techniques for hadronic decay modes not directly employable. In addition, at the high energy and luminosity projected at a future hadron collider, there will be numerous sources for contamination including initial- and final-state radiation, underlying event, or pile-up which must be mitigated. We propose a simple strategy to tag such "hyper-boosted" objects that defines jets with radii that scale inversely proportional to their transverse boost and combines the standard calorimetric information with charged track-based observables. By means of a fast detector simulation, we apply it to top quark identification and demonstrate that our method efficiently discriminates hadronically decaying top quarks from light QCD jets up to transverse boosts of 20 TeV. Our results open the way to tagging heavy objects with energies in the multi-TeV range at present and future hadron colliders.

  16. Visual tracking by separability-maximum boosting

    NASA Astrophysics Data System (ADS)

    Hou, Jie; Mao, Yao-bin; Sun, Jin-sheng

    2013-10-01

    Recently, visual tracking has been formulated as a classification problem whose task is to detect the object from the scene with a binary classifier. Boosting based online feature selection methods, which adopt the classifier to appearance changes by choosing the most discriminative features, have been demonstrated to be effective for visual tracking. A major problem of such online feature selection methods is that an inaccurate classifier may give imprecise tracking windows. Tracking error accumulates when the tracker trains the classifier with misaligned samples and finally leads to drifting. Separability-maximum boosting (SMBoost), an alternative form of AdaBoost which characterizes the separability between the object and the scene by their means and covariance matrices, is proposed. SMBoost only needs the means and covariance matrices during training and can be easily adopted to online learning problems by estimating the statistics incrementally. Experiment on UCI machine learning datasets shows that SMBoost is as accurate as offline AdaBoost, and significantly outperforms Oza's online boosting. Accurate classifier stabilizes the tracker on challenging video sequences. Empirical results also demonstrate improvements in term of tracking precision and speed, comparing ours to those state-of-the-art ones.

  17. DNA prime–protein boost increased the titer, avidity and persistence of anti-Aβ antibodies in wild-type mice

    PubMed Central

    Davtyan, H; Mkrtichyan, M; Movsesyan, N; Petrushina, I; Mamikonyan, G; Cribbs, DH; Agadjanyan, MG; Ghochikyan, A

    2010-01-01

    Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-β (Aβ42) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Aβ immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide–protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Aβ11 immune responses by developing an improved DNA vaccination protocol of the prime–boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Aβ antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime–boost regimen were long-lasting and possessed a higher avidity for binding with an Aβ42 peptide. Thus, we showed that a heterologous prime–boost regimen could be an effective protocol for developing a potent Alzheimer’s disease (AD) vaccine. PMID:19865176

  18. 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

  19. Centrifugal compressor design for electrically assisted boost

    NASA Astrophysics Data System (ADS)

    Y Yang, M.; Martinez-Botas, R. F.; Zhuge, W. L.; Qureshi, U.; Richards, B.

    2013-12-01

    Electrically assisted boost is a prominent method to solve the issues of transient lag in turbocharger and remains an optimized operation condition for a compressor due to decoupling from turbine. Usually a centrifugal compressor for gasoline engine boosting is operated at high rotational speed which is beyond the ability of an electric motor in market. In this paper a centrifugal compressor with rotational speed as 120k RPM and pressure ratio as 2.0 is specially developed for electrically assisted boost. A centrifugal compressor including the impeller, vaneless diffuser and the volute is designed by meanline method followed by 3D detailed design. Then CFD method is employed to predict as well as analyse the performance of the design compressor. The results show that the pressure ratio and efficiency at design point is 2.07 and 78% specifically.

  20. 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

  1. A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

    PubMed

    Zhang, Peng; Du, Enqi; 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.

  2. 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

  3. 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

  4. Adenovirus urethritis and concurrent conjunctivitis: a case series and review of the literature.

    PubMed

    Liddle, Olivia Louise; Samuel, Mannampallil Itty; Sudhanva, Malur; Ellis, Joanna; Taylor, Chris

    2015-03-01

    We present eight cases and review the literature of concurrent urethritis and conjunctivitis where adenovirus was identified as the causative pathogen. The focus of this review concerns the identification of specific sexual practices, symptoms, signs and any serotypes that seem more commonly associated with such adenovirus infections. We discuss the seasonality of adenovirus infection and provide practical advice for clinicians to give to the patient.

  5. Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A

    PubMed Central

    Stylianou, E.; Griffiths, K.L.; Poyntz, H.C.; Harrington-Kandt, R.; Dicks, M.D.; Stockdale, L.; Betts, G.; McShane, H.

    2015-01-01

    A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further. PMID:26478198

  6. 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.

  7. 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

  8. 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

  9. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  10. 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.

  11. Novel bat adenoviruses with an extremely large E3 gene.

    PubMed

    Tan, Bing; Yang, Xing-Lou; Ge, Xing-Yi; Peng, Cheng; Zhang, Yun-Zhi; Zhang, Li-Biao; Shi, Zheng-Li

    2016-07-01

    Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs. PMID:27032099

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

    PubMed

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

    2013-06-01

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

  13. 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

  14. 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

  15. Reassessing culture media and critical metabolites that affect adenovirus production.

    PubMed

    Shen, Chun Fang; Voyer, Robert; Tom, Roseanne; Kamen, Amine

    2010-01-01

    Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.

  16. The Attentional Boost Effect and Context Memory

    ERIC Educational Resources Information Center

    Mulligan, Neil W.; Smith, S. Adam; Spataro, Pietro

    2016-01-01

    Stimuli co-occurring with targets in a detection task are better remembered than stimuli co-occurring with distractors--the attentional boost effect (ABE). The ABE is of interest because it is an exception to the usual finding that divided attention during encoding impairs memory. The effect has been demonstrated in tests of item memory but it is…

  17. Schools Enlisting Defense Industry to Boost STEM

    ERIC Educational Resources Information Center

    Trotter, Andrew

    2008-01-01

    Defense contractors Northrop Grumman Corp. and Lockheed Martin Corp. are joining forces in an innovative partnership to develop high-tech simulations to boost STEM--or science, technology, engineering, and mathematics--education in the Baltimore County schools. The Baltimore County partnership includes the local operations of two major military…

  18. Cleanouts boost Devonian shale gas flow

    SciTech Connect

    Not Available

    1991-02-04

    Cleaning shale debris from the well bores is an effective way to boost flow rates from old open hole Devonian shale gas wells, research on six West Virginia wells begun in 1985 has shown. Officials involved with the study say the Appalachian basin could see 20 year recoverable gas reserves hiked by 315 bcf if the process is used on a wide scale.

  19. Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice

    PubMed Central

    HE, LEI; GONG, HAN-XIAN; LI, XIANG-PEN; WANG, YI-DONG; LI, YI; HUANG, JUN-JIAN; XIE, DAN; KUNG, HSIANG-FU; PENG, YING

    2013-01-01

    A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumor effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma (HCC) was explored in vitro and in vivo to investigate the possible mechanisms. The results indicated that recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of the Hepa 1–6 HCC cells. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1–6 cells. HCC was inhibited significantly when a single dose of 5×107 pfu rAdv-hTERTC27 was administered intravenously. In summary, the results of this study demonstrated that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer. PMID:24137404

  20. Cis and trans activation of adenovirus IVa2 gene transcription.

    PubMed Central

    Natarajan, V; Salzman, N P

    1985-01-01

    The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter in front of a gene coding for bacterial chloramphenicol acetyl transferase (CATase) and estimating levels of CATase and IVa2 promoter specific RNA synthesized after transfection. To produce detectable amounts of CATase with the IVa2 promoter, an enhancer has to be present in cis. In the absence of enhancer sequences, the adenovirus E1A gene can not stimulate CATase synthesis. When cells were transfected with plasmids containing enhancer sequences and various IVa2 mutant promoters upstream of the CAT gene, we observed that CATase activity was not reduced significantly even after deletion of all sequences upstream of the RNA initiation site. Synthesis of IVa2 specific RNA was dependent on plasmids containing an enhancer (SV40 72 bp repeat) that was present in cis. In the absence of enhancer sequences, co-transfection to provide the adenovirus E1A gene in trans also stimulated IVa2 RNA synthesis. When HeLa cells were transfected with various deletion mutants with an enhancer in cis it was seen that sequences -38 to -64 base pairs upstream of the RNA initiation site are necessary for efficient transcription. The E1A gene in trans and an enhancer in cis have an additive effect on RNA synthesis from both IVa2 and major late promoters. The basis for the conflicting results between transcription and CATase synthesis is discussed. Images PMID:2989786

  1. 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

  2. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

    PubMed

    Régulier, E; Paul, S; Marigliano, M; Kintz, J; Poitevin, Y; Ledoux, C; Roecklin, D; Cauet, G; Calenda, V; Homann, H E

    2001-01-01

    Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors. PMID:11219493

  3. Ex vivo adenovirus-mediated gene transfer and immunomodulatory protein production in human cornea.

    PubMed

    Oral, H B; Larkin, D F; Fehervari, Z; Byrnes, A P; Rankin, A M; Haskard, D O; Wood, M J; Dallman, M J; George, A J

    1997-07-01

    One attractive strategy to prevent or control allograft rejection is to genetically modify the donor tissue before transplantation. In this study, we have examined the feasibility of gene transfer to human corneal endothelium, using a number of recombinant adenovirus constructs. Ex vivo infection of human corneas with adenoviral vectors containing lacZ, under transcriptional control of either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters, provided high-level gene expression, which was largely restricted to endothelium. Expression of the reporter gene persisted at relatively high levels for up to 7 days, followed by a decline to indetectable levels by 28 days. RT-PCR analysis of lacZ transcription showed a similar picture with a short period (3-7 days) of RNA transcription after infection. In contrast, adenoviral DNA persisted for at least 56 days. Subsequently, we examined the expression of a potential therapeutic gene, CTLA-4 Ig fusion protein. Following infection of human corneas with adenoviral vectors encoding CTLA-4 Ig protein, high levels of the fusion protein were detected in corneal culture supernatants for up to 28 days. This protein was functionally active, as determined by binding to B7.1 (CD80)-expressing transfectants. This study suggests that genetic alteration of donor cornea before transplantation is a feasible approach for preventing or controlling allograft rejection. Similar gene-based strategies might also be feasible to prevent rejection of other transplanted tissues or organs. PMID:9282165

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers.

    PubMed

    Sibley, Samuel D; Goldberg, Tony L; Pedersen, Joel A

    2011-07-01

    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.

  10. Discussion of "the evolution of boosting algorithms" and "extending statistical boosting".

    PubMed

    Bühlmann, P; Gertheiss, J; Hieke, S; Kneib, T; Ma, S; Schumacher, M; Tutz, G; Wang, C-Y; Wang, Z; Ziegler, A

    2014-01-01

    This article is part of a For-Discussion-Section of Methods of Information in Medicine about the papers "The Evolution of Boosting Algorithms - From Machine Learning to Statistical Modelling" and "Extending Statistical Boosting - An Overview of Recent Methodological Developments", written by Andreas Mayr and co-authors. It is introduced by an editorial. This article contains the combined commentaries invited to independently comment on the Mayr et al. papers. In subsequent issues the discussion can continue through letters to the editor.

  11. 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...

  12. 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

  13. Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor

    PubMed Central

    Arnberg, Niklas; Edlund, Karin; Kidd, Alistair H.; Wadell, Göran

    2000-01-01

    Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2. PMID:10590089

  14. 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

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

    PubMed

    Gallaher, Sean D; Berk, Arnold J

    2013-09-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called "infectious genome titration" in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity.

  16. 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…

  17. S2 expressed from recombinant virus confers broad protection against IBV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that chickens primed with a recombinant LaSota virus (rLS) expressing the IBV S2 gene (rLS/IBV.S2) and boosted with an attenuated IBV Massachusetts (Mass)-type vaccine were protected against heterologous IBV Arkansas (Ark)-type virulent challenge. In the current study, we...

  18. S2 expressed from recombinant virus confers broad protection against infectious bronchitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that overexposing the IBV (infectious bronchitis virus) S2 to the chicken immune system by means of a vectored vaccine, followed by boost with whole virus, protects chickens against IBV showing dissimilar S1. We developed recombinant Newcastle disease virus (NDV) LaSota (...

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

    PubMed Central

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

    2014-01-01

    Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest. PMID:25486282

  20. Hemorrhagic enteritis by adenovirus-like particles in turkeys: a possible pathogenic mechanism.

    PubMed

    Gómez-Villamandos, J C; Carranza, J; Sierra, M A; Carrasco, L; Hervás, J; Blanco, A; Fernández, A

    1994-01-01

    This paper describes an outbreak of hemorrhagic enteritis due to adenovirus in turkeys in Spain. Diagnosis of the disease was confirmed by histopathological examination and the observation of adenovirus in spleen mononuclear cells and intestinal infiltrate. Evidence was also found of intravascular coagulation, which may give rise to the bleeding considered characteristic of this disease.

  1. 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.

  2. 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.

  3. Adenovirus Type 7 Pneumonia in Children Who Died from Measles-Associated Pneumonia, Hanoi, Vietnam, 2014.

    PubMed

    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; Nakajima, Noriko

    2016-04-01

    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

  4. Comparison of throat swab and nasopharyngeal aspirate specimens for rapid detection of adenovirus.

    PubMed

    Hara, Michimaru; Takao, Shinichi; Shimazu, Yukie

    2015-06-01

    Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.

  5. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    PubMed

    Muthumani, Kar; Wise, Megan C; Broderick, Kate E; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y; Weiner, David B

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  6. HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

    PubMed Central

    Muthumani, Kar; Wise, Megan C.; Broderick, Kate E.; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B.; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y.; Weiner, David B.

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  7. 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.

  8. Bioactive Molecule Prediction Using Extreme Gradient Boosting.

    PubMed

    Babajide Mustapha, Ismail; Saeed, Faisal

    2016-01-01

    Following the explosive growth in chemical and biological data, the shift from traditional methods of drug discovery to computer-aided means has made data mining and machine learning methods integral parts of today's drug discovery process. In this paper, extreme gradient boosting (Xgboost), which is an ensemble of Classification and Regression Tree (CART) and a variant of the Gradient Boosting Machine, was investigated for the prediction of biological activity based on quantitative description of the compound's molecular structure. Seven datasets, well known in the literature were used in this paper and experimental results show that Xgboost can outperform machine learning algorithms like Random Forest (RF), Support Vector Machines (LSVM), Radial Basis Function Neural Network (RBFN) and Naïve Bayes (NB) for the prediction of biological activities. In addition to its ability to detect minority activity classes in highly imbalanced datasets, it showed remarkable performance on both high and low diversity datasets. PMID:27483216

  9. Dendritic Cells Transduced with an Adenovirus Vector Encoding Epstein-Barr Virus Latent Membrane Protein 2B: a New Modality for Vaccination

    PubMed Central

    Ranieri, E.; Herr, W.; Gambotto, A.; Olson, W.; Rowe, D.; Robbins, P. D.; Kierstead, L. Salvucci; Watkins, S. C.; Gesualdo, L.; Storkus, W. J.

    1999-01-01

    Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8+ T lymphocytes from HLA-A2.1+, EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8+ T cells. Responder CD8+ T cells were tested for gamma interferon (IFN-γ) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-γ spot formation (T-cell frequency, <0.003%). IFN-γ ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8+ effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies. PMID:10559360

  10. A Defined Tuberculosis Vaccine Candidate Boosts BCG and Protects Against Multidrug Resistant Mycobacterium tuberculosis

    PubMed Central

    Bertholet, Sylvie; Ireton, Gregory C.; Ordway, Diane J.; Windish, Hillarie Plessner; Pine, Samuel O.; Kahn, Maria; Phan, Tony; Orme, Ian M.; Vedvick, Thomas S.; Baldwin, Susan L.; Coler, Rhea N.; Reed, Steven G.

    2011-01-01

    Despite the widespread use of Mycobacterium bovis bacillus Calmette-Guerin (BCG) childhood vaccine, tuberculosis (TB) remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG will include antigens that induce or recall appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens, including members of the virulence factor families PE/PPE and EsX, or antigens associated with latency were produced as a single recombinant fusion protein. When administered with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein ID93 was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, ID93/GLA-SE combination induced polyfunctional CD4 TH1-cell responses characterized by antigen-specific IFN-gamma, tumor necrosis factor and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals subsequently infected with virulent or multidrug resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with ID93/GLA-SE resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, ID93 elicited polyfunctional effector CD4 and CD8 T-cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine ID93/GLA-SE protects against TB and MDR-TB in animals, and is a candidate for boosting the protective efficacy of the childhood BCG vaccine. PMID:20944089

  11. Voltage-Boosting Driver For Switching Regulator

    NASA Technical Reports Server (NTRS)

    Trump, Ronald C.

    1990-01-01

    Driver circuit assures availability of 10- to 15-V gate-to-source voltage needed to turn on n-channel metal oxide/semiconductor field-effect transistor (MOSFET) acting as switch in switching voltage regulator. Includes voltage-boosting circuit efficiently providing gate voltage 10 to 15 V above supply voltage. Contains no exotic parts and does not require additional power supply. Consists of NAND gate and dual voltage booster operating in conjunction with pulse-width modulator part of regulator.

  12. Image enhancement based on edge boosting algorithm

    NASA Astrophysics Data System (ADS)

    Ngernplubpla, Jaturon; Chitsobhuk, Orachat

    2015-12-01

    In this paper, a technique for image enhancement based on proposed edge boosting algorithm to reconstruct high quality image from a single low resolution image is described. The difficulty in single-image super-resolution is that the generic image priors resided in the low resolution input image may not be sufficient to generate the effective solutions. In order to achieve a success in super-resolution reconstruction, efficient prior knowledge should be estimated. The statistics of gradient priors in terms of priority map based on separable gradient estimation, maximum likelihood edge estimation, and local variance are introduced. The proposed edge boosting algorithm takes advantages of these gradient statistics to select the appropriate enhancement weights. The larger weights are applied to the higher frequency details while the low frequency details are smoothed. From the experimental results, the significant performance improvement quantitatively and perceptually is illustrated. It can be seen that the proposed edge boosting algorithm demonstrates high quality results with fewer artifacts, sharper edges, superior texture areas, and finer detail with low noise.

  13. Exposure fusion using boosting Laplacian pyramid.

    PubMed

    Shen, Jianbing; Zhao, Ying; Yan, Shuicheng; Li, Xuelong

    2014-09-01

    This paper proposes a new exposure fusion approach for producing a high quality image result from multiple exposure images. Based on the local weight and global weight by considering the exposure quality measurement between different exposure images, and the just noticeable distortion-based saliency weight, a novel hybrid exposure weight measurement is developed. This new hybrid weight is guided not only by a single image's exposure level but also by the relative exposure level between different exposure images. The core of the approach is our novel boosting Laplacian pyramid, which is based on the structure of boosting the detail and base signal, respectively, and the boosting process is guided by the proposed exposure weight. Our approach can effectively blend the multiple exposure images for static scenes while preserving both color appearance and texture structure. Our experimental results demonstrate that the proposed approach successfully produces visually pleasing exposure fusion images with better color appearance and more texture details than the existing exposure fusion techniques and tone mapping operators. PMID:25137687

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

    PubMed Central

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

    1996-01-01

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

  15. 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

  16. Characterization of a New Species of Adenovirus in Falcons

    PubMed Central

    Schrenzel, Mark; Oaks, J. Lindsay; Rotstein, Dave; Maalouf, Gabriel; Snook, Eric; Sandfort, Cal; Rideout, Bruce

    2005-01-01

    In 1996, a disease outbreak occurred at a captive breeding facility in Idaho, causing anorexia, dehydration, and diarrhea or sudden death in 72 of 110 Northern aplomado falcons (Falco femoralis septentrionalis) from 9 to 35 days of age and in 6 of 102 peregrine falcons (Falco peregrinus) from 14 to 25 days of age. Sixty-two Northern aplomado and six peregrine falcons died. Epidemiologic analyses indicated a point source epizootic, horizontal transmission, and increased relative risk associated with cross-species brooding of eggs. Primary lesions in affected birds were inclusion body hepatitis, splenomegaly, and enteritis. The etiology in all mortalities was determined by molecular analyses to be a new species of adenovirus distantly related to the group I avian viruses, serotypes 1 and 4, Aviadenovirus. In situ hybridization and PCR demonstrated that the virus was epitheliotropic and lymphotropic and that infection was systemic in the majority of animals. Adeno-associated virus was also detected by PCR in most affected falcons, but no other infectious agents or predisposing factors were found in any birds. Subsequent to the 1996 epizootic, a similar disease caused by the same adenovirus was found over a 5-year period in orange-breasted falcons (Falco deiroleucus), teita falcons (Falco fasciinucha), a merlin (Falco columbarius), a Vanuatu peregrine falcon (Falco peregrinus nesiotes), and gyrfalcon × peregrine falcon hybrids (Falco rusticolus/peregrinus) that died in Wyoming, Oklahoma, Minnesota, and California. These findings indicate that this newly recognized adenovirus is widespread in western and midwestern North America and can be a primary pathogen in different falcon species. PMID:16000466

  17. Interactions of human lacrimal and salivary cystatins with adenovirus endopeptidase.

    PubMed

    Ruzindana-Umunyana, A; Weber, J M

    2001-09-01

    Over 100 serotypes of adenoviruses have been implicated in a variety of human and domesticated animal pathologies and some serotypes are widely used as gene transfer vectors. Aside from the limited use of vaccines for specific serotypes, little effort has been expended in the development of antivirals. The objective here was to study the effect of cystatins from human saliva (CS) and tears (CT), two points of viral entry, on adenain, the adenovirus type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both fluids at a yield of 5 mg/l. In vitro adenain activity was inhibited to 50% at a molar ratio of 5 CS:1 adenain and 3 CT:1 adenain. By comparison, papain was inhibited to 50% at a molar ratio of 2 CS:1 papain and 1.5 CT:1 papain. Adenain differed from papain in response to CS and chicken egg white (CEW) cystatin in being stimulated at low concentrations, and in being inhibited only at very high concentrations of cystatins. The presence of cleavage consensus sites specific to adenain in the human cystatins could drive the adenain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denaturation and by highly active fresh enzyme preparations. Our experiments designed to test the nature of the interaction between adenain and cystatins suggest a docking model for the adenain-human cystatin interaction, similar to that proposed for papain and CEW. At equilibrium the dissociation constant, K(d), between adenain and CT was 1.2 nM. The kinetic parameters determined here suggest a simple reversible mechanism for the inhibition of adenain by human cystatins. We conclude that the cystatins present in tears and saliva are unlikely to play a significant role in inhibiting adenovirus infections.

  18. Characterization of a new species of adenovirus in falcons.

    PubMed

    Schrenzel, Mark; Oaks, J Lindsay; Rotstein, Dave; Maalouf, Gabriel; Snook, Eric; Sandfort, Cal; Rideout, Bruce

    2005-07-01

    In 1996, a disease outbreak occurred at a captive breeding facility in Idaho, causing anorexia, dehydration, and diarrhea or sudden death in 72 of 110 Northern aplomado falcons (Falco femoralis septentrionalis) from 9 to 35 days of age and in 6 of 102 peregrine falcons (Falco peregrinus) from 14 to 25 days of age. Sixty-two Northern aplomado and six peregrine falcons died. Epidemiologic analyses indicated a point source epizootic, horizontal transmission, and increased relative risk associated with cross-species brooding of eggs. Primary lesions in affected birds were inclusion body hepatitis, splenomegaly, and enteritis. The etiology in all mortalities was determined by molecular analyses to be a new species of adenovirus distantly related to the group I avian viruses, serotypes 1 and 4, Aviadenovirus. In situ hybridization and PCR demonstrated that the virus was epitheliotropic and lymphotropic and that infection was systemic in the majority of animals. Adeno-associated virus was also detected by PCR in most affected falcons, but no other infectious agents or predisposing factors were found in any birds. Subsequent to the 1996 epizootic, a similar disease caused by the same adenovirus was found over a 5-year period in orange-breasted falcons (Falco deiroleucus), teita falcons (Falco fasciinucha), a merlin (Falco columbarius), a Vanuatu peregrine falcon (Falco peregrinus nesiotes), and gyrfalcon x peregrine falcon hybrids (Falco rusticolus/peregrinus) that died in Wyoming, Oklahoma, Minnesota, and California. These findings indicate that this newly recognized adenovirus is widespread in western and midwestern North America and can be a primary pathogen in different falcon species.

  19. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  20. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  1. Transductional targeting of adenovirus vectors for gene therapy

    PubMed Central

    Glasgow, JN; Everts, M; Curiel, DT

    2007-01-01

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

  2. Human immunodeficiency virus type 1 envelope-specific cytotoxic T lymphocytes response dynamics after prime-boost vaccine regimens with human immunodeficiency virus type 1 canarypox and pseudovirions.

    PubMed

    Arp, J; Rovinski, B; Sambhara, S; Tartaglia, J; Dekaban, G

    1999-01-01

    Virus-specific cytotoxic T lymphocytes (CTLs) may represent significant immune mechanisms in the control of human immunodeficiency virus (HIV) infection and, therefore, CTL induction may be a fundamental goal in the development of an efficacious acquired immunodeficiency syndrome (AIDS) vaccine. In the current study, prime-boost protocols were used to investigate the potential of noninfectious human immunodeficiency virus type 1 (HIV-1) pseudovirions (HIV PSV) in enhancing HIV-specific CTL responses in Balb/c mice primed with the recombinant canarypox vector, vCP205, encoding HIV-1 gp120 (MN strain) in addition to Gag/Protease (HIB strain). The prime-boost immunization regimens were administered intramuscularly and involved injections of vCP205 followed by boosts with HIV PSV. Previous vaccination strategies solely involving vCP205 had induced good cellular immune responses in uninfected human volunteers, despite some limitations. The use of genetically engineered HIV PSV was a logical step in the evaluation of whole noninfectious virus or inactivated virus vaccine strategies, particularly as a potential boosting agent for vCP205-primed recipients. Based on this current study, HIV PSV appeared to have the capability to effectively induce and boost cell-mediated HIV-1-specific responses. In order to observe the immune effects of HIV PSV in a prime-boost immunization strategy, both HIV vaccine immunogens required careful titration in vivo. This suggests that careful consideration should be given to the optimization of immunization protocols destined for human use.

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

    PubMed Central

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

    2005-01-01

    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14–amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti–P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-γ–positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene β-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective. PMID:15841217

  4. [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

  5. 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

  6. Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy.

    PubMed

    Madisch, Ijad; Harste, Gabi; Pommer, Heidi; Heim, Albert

    2005-12-01

    Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.

  7. Phylogenetic Analysis of the Main Neutralization and Hemagglutination Determinants of All Human Adenovirus Prototypes as a Basis for Molecular Classification and Taxonomy

    PubMed Central

    Madisch, Ijad; Harste, Gabi; Pommer, Heidi; Heim, Albert

    2005-01-01

    Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization ɛ determinant (loops 1 and 2) and the hemagglutination γ determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. ɛ and γ determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of ɛ determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (γ determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (ɛ determinant) and a speculative, not-yet-isolated HAdV prototype (γ determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the ɛ determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, γ determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution. PMID:16306598

  8. 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.

  9. Biology of E1-Deleted Adenovirus Vectors in Nonhuman Primate Muscle

    PubMed Central

    Zoltick, Philip W.; Chirmule, Narendra; Schnell, Michael A.; Gao, Guang-ping; Hughes, Joseph V.; Wilson, James M.

    2001-01-01

    Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors. PMID:11333904

  10. 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.

  11. Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

    PubMed Central

    Stewart, P L; Chiu, C Y; Huang, S; Muir, T; Zhao, Y; Chait, B; Mathias, P; Nemerow, G R

    1997-01-01

    Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies. PMID:9135136

  12. Targeting the replication of adenovirus to p53-defective thyroid carcinoma with a p53-regulated Cre/loxP system.

    PubMed

    Nagayama, Y; Nishihara, E; Namba, H; Yokoi, H; Hasegawa, M; Mizuguchi, H; Hayakawa, T; Hamada, H; Yamashita, S; Niwa, M

    2001-01-01

    In this article, we evaluated the feasibility of the restricted replication-competent adenoviruses for treatment of anaplastic thyroid carcinomas (ATCs), which are very aggressive and difficult to treat. Because ATCs very often harbor p53 mutations, we used wt-p53 as a regulatory factor to restrict virus replication and cytopathic effect to p53-mutated cells. The recently reported "gene inactivation strategy" using p53-regulated Cre/loxP system was employed; this system consists of two recombinant adenoviruses. One has an expression unit of the synthetic p53 - responsive promoter and the Cre recombinase gene (Axyp53RECre), and another contains two expression units; the first consists of E1A gene flanked by a pair of loxP sites downstream of the constitutive CAG promoter and the second E1B19K gene under the control of the CMV promoter (AdCALE1AL). We expected that coinfection of these two adenoviruses into the cells with wt-p53 would lead to expression of the Cre, which excises E1A gene and switches off E1A expression resulting in no virus replication, whereas in the cells with mutant p53 E1A could be expressed that leads to virus replication and cell lysis. Our in vitro data demonstrate that although infection of AdCALE1AL alone led to E1A expression, viral replication and cytolysis in all the thyroid cells examined irrespective of their p53 status, the double infection did so in FRO cells (p53-null ATC) but not in FRO cells stably expressing wt-p53 and normal thyroid cells with wt-p53. These data indicate that our double infection method may have a potential for treatment of ATC and probably also other p53-defective cancer cells. PMID:11219492

  13. 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

  14. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla).

    PubMed

    Wevers, Diana; Leendertz, Fabian H; Scuda, Nelly; Boesch, Christophe; Robbins, Martha M; Head, Josephine; Ludwig, Carsten; Kühn, Joachim; Ehlers, Bernhard

    2010-11-05

    Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2-10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

  15. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

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

    2015-08-15

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

  16. 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

  17. Detection of adenovirus using PCR and molecular beacon.

    PubMed

    Poddar, S K

    1999-09-01

    The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.

  18. 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

  19. Hybrid Recovery-less Method Soft Switching Boost Chopper Circuit

    NASA Astrophysics Data System (ADS)

    Yamamoto, Masayoshi; Toda, Hirotaka; Kawashima, Takahiro; Yoshida, Toshiyuki

    The conventional recovery-less boost type converter cannot achieve the soft switching operation in case of the turn off transition. In this paper, the novel hybrid recovery-less boost type converter, which can achieve the soft switching turn off transition, is proposed. Furthermore, the proposed hybrid recovery-less boost type converter has the switch function between the conventional recovery-less mode and the proposed soft switching mode. In general, the efficiency in the soft switching converter is less than the hard switching in case of the lower output power condition. However, using the switch function of the proposed boost type converter, the hybrid recovery-less boost type converter can achieve the high efficiency performance in the whole output power area in spite of the soft switching operation. The proposed hybrid recovery-less boost type converter is evaluated and discussed from experimental point of view.

  20. Boost matrix converters in clean energy systems

    NASA Astrophysics Data System (ADS)

    Karaman, Ekrem

    This dissertation describes an investigation of novel power electronic converters, based on the ultra-sparse matrix topology and characterized by the minimum number of semiconductor switches. The Z-source, Quasi Z-source, Series Z-source and Switched-inductor Z-source networks were originally proposed for boosting the output voltage of power electronic inverters. These ideas were extended here on three-phase to three-phase and three-phase to single-phase indirect matrix converters. For the three-phase to three-phase matrix converters, the Z-source networks are placed between the three-switch input rectifier stage and the output six-switch inverter stage. A brief shoot-through state produces the voltage boost. An optimal pulse width modulation technique was developed to achieve high boosting capability and minimum switching losses in the converter. For the three-phase to single-phase matrix converters, those networks are placed similarly. For control purposes, a new modulation technique has been developed. As an example application, the proposed converters constitute a viable alternative to the existing solutions in residential wind-energy systems, where a low-voltage variable-speed generator feeds power to the higher-voltage fixed-frequency grid. Comprehensive analytical derivations and simulation results were carried out to investigate the operation of the proposed converters. Performance of the proposed converters was then compared between each other as well as with conventional converters. The operation of the converters was experimentally validated using a laboratory prototype.

  1. Boosting family income to promote child development.

    PubMed

    Duncan, Greg J; Magnuson, Katherine; Votruba-Drzal, Elizabeth

    2014-01-01

    Families who live in poverty face disadvantages that can hinder their children's development in many ways, write Greg Duncan, Katherine Magnuson, and Elizabeth Votruba-Drzal. As they struggle to get by economically, and as they cope with substandard housing, unsafe neighborhoods, and inadequate schools, poor families experience more stress in their daily lives than more affluent families do, with a host of psychological and developmental consequences. Poor families also lack the resources to invest in things like high-quality child care and enriched learning experiences that give more affluent children a leg up. Often, poor parents also lack the time that wealthier parents have to invest in their children, because poor parents are more likely to be raising children alone or to work nonstandard hours and have inflexible work schedules. Can increasing poor parents' incomes, independent of any other sort of assistance, help their children succeed in school and in life? The theoretical case is strong, and Duncan, Magnuson, and Votruba-Drzal find solid evidence that the answer is yes--children from poor families that see a boost in income do better in school and complete more years of schooling, for example. But if boosting poor parents' incomes can help their children, a crucial question remains: Does it matter when in a child's life the additional income appears? Developmental neurobiology strongly suggests that increased income should have the greatest effect during children's early years, when their brains and other systems are developing rapidly, though we need more evidence to prove this conclusively. The authors offer examples of how policy makers could incorporate the findings they present to create more effective programs for families living in poverty. And they conclude with a warning: if a boost in income can help poor children, then a drop in income--for example, through cuts to social safety net programs like food stamps--can surely harm them.

  2. Boosting salt resistance of short antimicrobial peptides.

    PubMed

    Chu, Hung-Lun; Yu, Hui-Yuan; Yip, Bak-Sau; Chih, Ya-Han; Liang, Chong-Wen; Cheng, Hsi-Tsung; Cheng, Jya-Wei

    2013-08-01

    The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, therefore limiting their use as pharmaceutical agents. Here, we describe a strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the nonnatural bulky amino acid β-naphthylalanine to their termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine end-tagged variants were less affected.

  3. Boost covariant gluon distributions in large nuclei

    NASA Astrophysics Data System (ADS)

    McLerran, Larry; Venugopalan, Raju

    1998-04-01

    It has been shown recently that there exist analytical solutions of the Yang-Mills equations for non-Abelian Weizsäcker-Williams fields which describe the distribution of gluons in large nuclei at small x. These solutions however depend on the color charge distribution at large rapidities. We here construct a model of the color charge distribution of partons in the fragmentation region and use it to compute the boost covariant momentum distributions of wee gluons. The phenomenological applications of our results are discussed.

  4. 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

  5. Synergistic tumor suppression by adenovirus-mediated ING4/PTEN double gene therapy for gastric cancer.

    PubMed

    Zhang, H; Zhou, X; Xu, C; Yang, J; Xiang, J; Tao, M; Xie, Y

    2016-01-01

    Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog (PTEN) have been shown to be strong candidate tumor suppressors. However, the combined efficacy of ING4 and PTEN for human gastric cancer remains to be determined. In this report, we constructed a multiple promoter expression cassette-based recombinant adenovirus coexpressing ING4 and PTEN (AdVING4/PTEN), assessed the combined effects of AdVING4/PTEN on gastric cancer using wild-type p53 AGS and SNU-1 human gastric cancer cell lines, and elucidated its underlying mechanisms. We found that AdVING4/PTEN-induced synergistic growth inhibition and apoptosis in vitro AGS or SNU-1 tumor cells and in vivo AGS xenografted tumors subcutaneously inoculated in athymic BALB/c nude mice. Mechanistically, AdVING4/PTEN exhibited an enhanced effect on upregulation of p53, Ac-p53 (K382), P21, Bax, PUMA, Noxa, cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP as well as downregulation of Bcl-2 in vitro and in vivo. In addition, AdVING4/PTEN synergistically downregulated tumor vessel CD34 expression and reduced microvessel density, and additively inhibited vascular endothelial growth factor (VEGF) expression in vivo. The synergistic tumor suppression elicited by AdVING4/PTEN was closely associated with the synergistic induction of apoptosis possibly via enhancement of endogenous p53 responses through cooperatively facilitating p53's stability and acetylation, and the synergistic inhibition of tumor angiogenesis probably via overlapping reduction of VEGF through cooperatively downregulating hypoxia inducible factor-1α's level and transcription activity. Thus, our results indicate that cancer gene therapy combining ING4 and PTEN may constitute a novel and effective therapeutic modality for human gastric cancer and other cancers.

  6. Adenovirus-based vaccine against Listeria monocytogenes: extending the concept of invariant chain linkage.

    PubMed

    Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Schlüter, Dirk; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2013-10-15

    The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further, vaccination of C57BL/6 (L. monocytogenes-resistant) and BALB/c (L. monocytogenes-susceptible) mice with adenoviral vectors encoding natural L. monocytogenes-derived soluble Ags (listeriolysin O and p60) revealed that tethering of these Ags to Ii markedly improved the vaccine-induced CD8(+) T cell response to two of three epitopes studied. More importantly, Ii linkage accelerated and augmented vaccine-induced protection in both mouse strains and prolonged protection, in particular that induced by the weak Ag, p60, in L. monocytogenes-susceptible BALB/c mice.

  7. 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

  8. Structure of Adenovirus Complexed with Its Internalization Receptor, αvβ5 Integrin

    PubMed Central

    Chiu, Charles Y.; Mathias, Patricia; Nemerow, Glen R.; Stewart, Phoebe L.

    1999-01-01

    The three-dimensional structure of soluble recombinant integrin αvβ5 bound to human adenovirus types 2 and 12 (Ad2 and -12) has been determined at ∼21-Å resolution by cryoelectron microscopy (cryo-EM). The αvβ5 integrin is known to promote Ad cell entry. Cryo-EM has shown that the integrin-binding RGD (Arg-Gly-Asp) protrusion of the Ad2 penton base protein is highly mobile (P. L. Stewart, C. Y. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. R. Nemerow, EMBO J. 16:1189–1198, 1997). Sequence analysis indicated that the Ad12 RGD surface loop is shorter than that of Ad2 and probably less flexible, hence more suitable for structural characterization of the Ad-integrin complex. The cryo-EM structures of the two virus-receptor complexes revealed a ring of integrin density above the penton base of each virus serotype. As expected, the integrin density in the Ad2 complex was diffuse while that in the Ad12 complex was better defined. The integrin consists of two discrete subdomains, a globular domain with an RGD-binding cleft ∼20 Å in diameter and a distal domain with extended, flexible tails. Kinetic analysis of Ad2 interactions with αvβ5 indicated ∼4.2 integrin molecules bound per penton base at close to saturation. These results suggest that the precise spatial arrangement of five RGD protrusions on the penton base promotes integrin clustering and the signaling events required for virus internalization. PMID:10400774

  9. 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

  10. Delineation of interfaces on human alpha-defensins critical for human adenovirus and human papillomavirus inhibition.

    PubMed

    Tenge, Victoria R; Gounder, Anshu P; Wiens, Mayim E; Lu, Wuyuan; Smith, Jason G

    2014-09-01

    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.

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

    PubMed

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

    2016-01-01

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

  12. Recombinant baculovirus isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2007-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac or BaculoDirect, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  13. 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.

  14. A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells.

    PubMed

    Shayakhmetov, Dmitry M; Carlson, Cheryl A; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André

    2002-02-01

    To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.

  15. Effect of marker distance and orientation on recombinant formation in poxvirus-infected cells.

    PubMed Central

    Parks, R J; Evans, D H

    1991-01-01

    Little is known about the mechanism of poxvirus recombination even though construction of recombinant viruses by recombination-dependent methods is a widely adopted technique. We have shown previously that transfected DNAs are efficiently recombined while replicating in cells infected with Shope fibroma virus. Because recombinant DNA can be recovered from infected cells as a high-molecular-weight head-to-tail concatemer, it was possible to transfect genetically marked lambda DNAs into infected cells and assay recombinants as bacteriophage particles following in vitro packaging. This approach was used in this study to examine how marker distance and marker orientation influence recombination in Shope fibroma virus-infected cells. Simple two-factor crosses were readily modelled by using a mapping function derived from classical phage studies and showed low negative interference (I = -2.8 +/- 0.5) in crosses involving markers greater than 100 bp apart. More complex four- and five-factor crosses showed that the recombination frequency per unit distance was not constant (rising as the marker separation was reduced from 100 to 1 bp) and that crosses performed in poxvirus-infected cells are subject to high negative interference. One consequence is that marker orientation does not dramatically influence the outcome of most Shope fibroma virus-catalyzed crosses in clear contrast to what is observed in adenovirus or simian virus 40-infected cells. These results can be interpreted to indicate that similar statistical and physical constraints influence both viral and phage recombination and suggest that heteroduplexes may be important intermediates in the poxvirus recombination process. PMID:1847453

  16. Low temperature operation of a boost converter

    SciTech Connect

    Moss, B.S.; Boudreaux, R.R.; Nelms, R.M.

    1996-12-31

    The development of satellite power systems capable of operating at low temperatures on the order of 77K would reduce the heating system required on deep space vehicles. The power supplies in the satellite power system must be capable of operating at these temperatures. This paper presents the results of a study into the operation of a boost converter at temperatures close to 77K. The boost converter is designed to supply an output voltage and power of 42 V and 50 W from a 28 V input source. The entire system, except the 28 V source, is placed in the environmental chamber. This is important because the system does not require any manual adjustments to maintain a constant output voltage with a high efficiency. The constant 42 V output of this converter is a benefit of the application of a CMOS microcontroller in the feedback path. The switch duty cycle is adjusted by the microcontroller to maintain a constant output voltage. The efficiency of the system varied less than 1% over the temperature range of 22 C to {minus}184 C and was approximately 94.2% when the temperature was {minus}184 C.

  17. Domain adaptive boosting method and its applications

    NASA Astrophysics Data System (ADS)

    Geng, Jie; Miao, Zhenjiang

    2015-03-01

    Differences of data distributions widely exist among datasets, i.e., domains. For many pattern recognition, nature language processing, and content-based analysis systems, a decrease in performance caused by the domain differences between the training and testing datasets is still a notable problem. We propose a domain adaptation method called domain adaptive boosting (DAB). It is based on the AdaBoost approach with extensions to cover the domain differences between the source and target domains. Two main stages are contained in this approach: source-domain clustering and source-domain sample selection. By iteratively adding the selected training samples from the source domain, the discrimination model is able to achieve better domain adaptation performance based on a small validation set. The DAB algorithm is suitable for the domains with large scale samples and easy to extend for multisource adaptation. We implement this method on three computer vision systems: the skin detection model in single images, the video concept detection model, and the object classification model. In the experiments, we compare the performances of several commonly used methods and the proposed DAB. Under most situations, the DAB is superior.

  18. Jet substructures of boosted polarized top quarks

    NASA Astrophysics Data System (ADS)

    Kitadono, Yoshio; Li, Hsiang-nan

    2014-06-01

    We study jet substructures of a boosted polarized top quark, which undergoes the semileptonic decay t→bℓν, in the perturbative QCD framework. The jet mass distribution (energy profile) is factorized into the convolution of a hard top-quark decay kernel with the bottom-quark jet function (jet energy function). Computing the hard kernel to the leading order in QCD and inputting the latter functions from the resummation formalism, we observe that the jet mass distribution is not sensitive to the helicity of the top quark, but the energy profile is: energy is accumulated faster within a left-hand top jet than within a right-hand one, a feature related to the V-A structure of weak interaction. It is pointed out that the energy profile is a simple and useful jet observable for helicity discrimination of a boosted top quark, which helps identification of physics beyond the standard model at the Large Hadron Collider. The extension of our analysis to other jet substructures, including those associated with a hadronically decaying polarized top quark, is proposed.

  19. Brain glucosamine boosts protective glucoprivic feeding.

    PubMed

    Osundiji, Mayowa A; Zhou, Ligang; Shaw, Jill; Moore, Stephen P; Yueh, Chen-Yu; Sherwin, Robert; Heisler, Lora K; Evans, Mark L

    2010-04-01

    The risk of iatrogenic hypoglycemia is increased in diabetic patients who lose defensive glucoregulatory responses, including the important warning symptom of hunger. Protective hunger symptoms during hypoglycemia may be triggered by hypothalamic glucose-sensing neurons by monitoring changes downstream of glucose phosphorylation by the specialized glucose-sensing hexokinase, glucokinase (GK), during metabolism. Here we investigated the effects of intracerebroventricular (ICV) infusion of glucosamine (GSN), a GK inhibitor, on food intake at normoglycemia and protective feeding responses during glucoprivation and hypoglycemia in chronically catheterized rats. ICV infusion of either GSN or mannoheptulose, a structurally different GK inhibitor, dose-dependently stimulated feeding at normoglycemia. Consistent with an effect of GSN to inhibit competitively glucose metabolism, ICV coinfusion of d-glucose but not l-glucose abrogated the orexigenic effect of ICV GSN at normoglycemia. Importantly, ICV infusion of a low GSN dose (15 nmol/min) that was nonorexigenic at normoglycemia boosted feeding responses to glucoprivation in rats with impaired glucose counterregulation. ICV infusion of 15 nmol/min GSN also boosted feeding responses to threatened hypoglycemia in rats with defective glucose counterregulation. Altogether our findings suggest that GSN may be a potential therapeutic candidate for enhancing defensive hunger symptoms during hypoglycemia.

  20. Titration of adenovirus by counting cells containing virus-induced inclusion bodies.

    PubMed

    Weber, J

    1972-05-01

    A new method for the titration of adenovirus types 2 and 12 based on the enumeration of viral inclusions in infected cells was devised and evaluated. The technique gave virus titers comparable to those obtained by the plaque assay procedure.

  1. Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

    PubMed

    Walters, Robert W; Freimuth, Paul; Moninger, Thomas O; Ganske, Ingrid; Zabner, Joseph; Welsh, Michael J

    2002-09-20

    Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

  2. 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.

  3. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    PubMed

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  4. 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

  5. Fatal pulmonary edema in white-tailed deer (Odocoileus virginianus) associated with adenovirus infection.

    PubMed

    Sorden, S D; Woods, L W; Lehmkuhl, H D

    2000-07-01

    Sporadic sudden deaths in adult white-tailed deer occurred from November 1997 through August 1998 on an Iowa game farm. Three of the 4 deer necropsied had severe pulmonary edema, widespread mild lymphocytic vasculitis, and amphophilic intranuclear inclusion bodies in scattered endothelial cells in blood vessels in the lung and abdominal viscera. Immunohistochemistry with bovine adenovirus 5 antisera and transmission electron microscopy demonstrated adenoviral antigen and nucleocapsids, respectively, within endothelial cells. Adenovirus was isolated in cell culture from 1 of the affected deer. The isolate was neutralized by California black-tailed deer adenovirus antiserum. These findings indicate that adenovirus should be considered in the differential diagnosis of both black-tailed and white-tailed deer with pulmonary edema and/or hemorrhagic enteropathy.

  6. Quantitative detection of human adenoviruses in wastewater and combined sewer overflows influencing a Michigan river.

    PubMed

    Fong, Theng-Theng; Phanikumar, Mantha S; Xagoraraki, Irene; Rose, Joan B

    2010-02-01

    Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (n = 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 x 10(6) viruses/liter and 5.35 x 10(5) viruses/liter, respectively. Adenovirus removal was <2 log(10) (99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (n = 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 x 10(3) viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.

  7. Transgene delivery to cultured keratinocytes via replication-deficient adenovirus vectors.

    PubMed

    Ramirez, Vincent P; Aneskievich, Brian J

    2014-01-01

    Transient transgene expression can facilitate investigation of that gene-product function or effect on keratinocyte biology. Several chemical and biologic delivery systems are available, and among them adenoviruses offer particular advantages in efficiency and transgene capacity. Here we describe the advantages of bicistronic adenovirus and inclusion of the polycation hexadimethrine bromide to aid in the detection of positively transduced cells and enhance transduction efficiency. PMID:24281865

  8. [Experimental gene therapy using p21/WAF1 gene in esophageal squamous cell carcinoma--adenovirus infection and gene gun technology].

    PubMed

    Fujii, T; Tanaka, Y; Tanaka, T; Matono, S; Sueyoshi, S; Fujita, H; Shirouzu, K; Kato, S; Yamana, H

    2001-10-01

    p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

  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. 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

  11. Current issues and future directions of oncolytic adenoviruses.

    PubMed

    Yamamoto, Masato; Curiel, David T

    2010-02-01

    Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy.

  12. Adenovirus type 2 nuclear RNA accumulating during productive infection.

    PubMed Central

    Bachenheimer, S L

    1977-01-01

    The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus-type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Later nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and less than 32S after denaturation and a second having a narrow size distribution around 35S which is quite stable to Me2SO. Analysis of late RNA by hybridization to Sma fragments of Ad2 DNA suggests that the 35S RNA species is derived from a limited portion of the left half of the viral genome. PMID:864839

  13. Current Issues and Future Directions of Oncolytic Adenoviruses

    PubMed Central

    Yamamoto, Masato; Curiel, David T

    2009-01-01

    Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy. PMID:19935777

  14. 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

  15. Adenovirus Membrane Penetration: Tickling the Tail of a Sleeping Dragon

    PubMed Central

    Wiethoff, Christopher M.; Nemerow, Glen R.

    2015-01-01

    As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (1), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus(HAdV). PMID:25798531

  16. 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

  17. [Is there a risk of zoonotic disease due to adenoviruses?].

    PubMed

    Loustalot, Fabien; Creyssels, Sophie; Salinas, Sara; Benkõ, Mária; Harrach, Balázs; Mennechet, Franck J D; Kremer, Eric J

    2015-12-01

    Every year brings another round of zoonotic viral infections. Usually they fall under the radar, but the occasional lethal epidemic brings another scare to the public and new urgency to the medical community. The types of these viruses (DNA vs. RNA genomes, enveloped vs. proteinaceous) as well as the preceding host(s) vary. Over the last 20 years, bats have been identified as an enigmatic carrier for several pathogens that have jumped the species barrier and infected humans. Factors that favour the emergence of zoonotic pathogens include the increasing overlap of the human and animal habitats, cultural activities, and the host reservoir. In this context, we asked whether bat and/or nonhuman primate adenoviruses are a risk for human health. PMID:26672663

  18. Adenovirus membrane penetration: Tickling the tail of a sleeping dragon.

    PubMed

    Wiethoff, Christopher M; Nemerow, Glen R

    2015-05-01

    As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (Moyer and Nemerow, 2011, Curr. Opin. Virol., 1, 44-49), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus (HAdV).

  19. Boost in radiotherapy: external beam sunset, brachytherapy sunrise

    PubMed Central

    2009-01-01

    Radiobiological limitations for dose escalation in external radiotherapy are presented. Biological and clinical concept of brachytherapy boost to increase treatment efficacy is discussed, and different methods are compared. Oncentra Prostate 3D conformal real-time ultrasound-guided brachytherapy is presented as a solution for boost or sole therapy.

  20. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  1. 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.

  2. CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells

    PubMed Central

    Wang, Yaohe; Gangeswaran, Rathi; Zhao, Xingbo; Wang, Pengju; Tysome, James; Bhakta, Vipul; Yuan, Ming; Chikkanna-Gowda, C.P.; Jiang, Guozhong; Gao, Dongling; Cao, Fengyu; Francis, Jennelle; Yu, Jinxia; Liu, Kangdong; Yang, Hongyan; Zhang, Yunhan; Zang, Weidong; Chelala, Claude; Dong, Ziming; Lemoine, Nick

    2009-01-01

    The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics. PMID:19411761

  3. 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.

  4. Comparative investigations for adenovirus recognition and quantification: Plastic or natural antibodies?

    PubMed

    Altintas, Zeynep; Pocock, Jack; Thompson, Katy-Anne; Tothill, Ibtisam E

    2015-12-15

    Comparative and comprehensive investigations for adenovirus recognition and detection were conducted using plastic and natural antibodies to compare three different strategies. The implementation of molecularly imprinted polymer (MIP) technology for specific and sensitive recognition of viruses with the combination of biosensors was reported. Plastic antibodies (MIPs nanoparticles) were produced for adenovirus by employing a novel solid phase synthesis method. MIP receptors were then characterised using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques prior to immobilisation on a surface plasmon resonance (SPR) sensor as affinity receptor for adenovirus detection. Two different templates were also imprinted as control MIPs (vancomycin-MIP and MS2-MIP). The specific recognition of adenovirus was investigated in the concentration range of 0.01-20 pM and the limit of detection was achieved as 0.02 pM. As an alternative to MIP receptors, direct and sandwich assays were developed for adenovirus quantification using natural antibodies. The detection limit of direct and sandwich assays were found as 0.3 pM and 0.008 pM, respectively. The kinetic data analyses were performed for three different adenovirus recognition methods and cross-reactivity studies were also conducted using MS2 phage as control virus and an excellent specificity was achieved with all assays types. This work confirmed the suitability of the MIPs SPR sensor for the detection of viruses. PMID:26264266

  5. Particle Tracking of Intracellular Trafficking of Octaarginine-modified Liposomes: A Comparative Study With Adenovirus

    PubMed Central

    Akita, Hidetaka; Enoto, Kaoru; Masuda, Tomoya; Mizuguchi, Hiroyuki; Tani, Tomomi; Harashima, Hideyoshi

    2010-01-01

    It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)–tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips. PMID:20216528

  6. 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

  7. 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.

  8. [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.

  9. 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

  10. Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

    PubMed

    Nalbantoglu, J; Pari, G; Karpati, G; Holland, P C

    1999-04-10

    Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

  11. Boosting jet power in black hole spacetimes

    PubMed Central

    Neilsen, David; Lehner, Luis; Palenzuela, Carlos; Hirschmann, Eric W.; Liebling, Steven L.; Motl, Patrick M.; Garrett, Travis

    2011-01-01

    The extraction of rotational energy from a spinning black hole via the Blandford–Znajek mechanism has long been understood as an important component in models to explain energetic jets from compact astrophysical sources. Here we show more generally that the kinetic energy of the black hole, both rotational and translational, can be tapped, thereby producing even more luminous jets powered by the interaction of the black hole with its surrounding plasma. We study the resulting Poynting jet that arises from single boosted black holes and binary black hole systems. In the latter case, we find that increasing the orbital angular momenta of the system and/or the spins of the individual black holes results in an enhanced Poynting flux. PMID:21768341

  12. Hydrodynamic approach to boost invariant free streaming

    NASA Astrophysics Data System (ADS)

    Calzetta, E.

    2015-08-01

    We consider a family of exact boost invariant solutions of the transport equation for free-streaming massless particles, where the one-particle distribution function is defined in terms of a function of a single variable. The evolution of second and third moments of the one-particle distribution function [the second moment being the energy momentum tensor (EMT) and the third moment the nonequilibrium current (NEC)] depends only on two moments of that function. Given those two moments, we show how to build a nonlinear hydrodynamic theory which reproduces the early time evolution of the EMT and the NEC. The structure of these theories may give insight on nonlinear hydrodynamic phenomena on short time scales.

  13. Boosting low-mass hadronic resonances

    NASA Astrophysics Data System (ADS)

    Shimmin, Chase; Whiteson, Daniel

    2016-09-01

    Searches for new hadronic resonances typically focus on high-mass spectra due to overwhelming QCD backgrounds and detector trigger rates. We present a study of searches for relatively low-mass hadronic resonances at the LHC in the case that the resonance is boosted by recoiling against a well-measured high-pT probe such as a muon, photon or jet. The hadronic decay of the resonance is then reconstructed either as a single large-radius jet or as a resolved pair of standard narrow-radius jets, balanced in transverse momentum to the probe. We show that the existing 2015 LHC data set of p p collisions with ∫L d t =4 fb-1 should already have powerful sensitivity to a generic Z' model which couples only to quarks, for Z' masses ranging from 20 - 500 GeV /c2 .

  14. Boosted X Waves in Nonlinear Optical Systems

    SciTech Connect

    Arevalo, Edward

    2010-01-15

    X waves are spatiotemporal optical waves with intriguing superluminal and subluminal characteristics. Here we theoretically show that for a given initial carrier frequency of the system localized waves with genuine superluminal or subluminal group velocity can emerge from initial X waves in nonlinear optical systems with normal group velocity dispersion. Moreover, we show that this temporal behavior depends on the wave detuning from the carrier frequency of the system and not on the particular X-wave biconical form. A spatial counterpart of this behavior is also found when initial X waves are boosted in the plane transverse to the direction of propagation, so a fully spatiotemporal motion of localized waves can be observed.

  15. Boosting jet power in black hole spacetimes.

    PubMed

    Neilsen, David; Lehner, Luis; Palenzuela, Carlos; Hirschmann, Eric W; Liebling, Steven L; Motl, Patrick M; Garrett, Travis

    2011-08-01

    The extraction of rotational energy from a spinning black hole via the Blandford-Znajek mechanism has long been understood as an important component in models to explain energetic jets from compact astrophysical sources. Here we show more generally that the kinetic energy of the black hole, both rotational and translational, can be tapped, thereby producing even more luminous jets powered by the interaction of the black hole with its surrounding plasma. We study the resulting Poynting jet that arises from single boosted black holes and binary black hole systems. In the latter case, we find that increasing the orbital angular momenta of the system and/or the spins of the individual black holes results in an enhanced Poynting flux.

  16. Boosted X waves in nonlinear optical systems.

    PubMed

    Arévalo, Edward