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Al, Qurashi Yasir Mohammed A. "Molecular typing of adenoviruses." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506268.
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Rodríguez, Eduardo. "Virion- and VAP-receptor recognition in the human adenovirus type 2 system." Lund : Lund University, 1998. http://catalog.hathitrust.org/api/volumes/oclc/68945080.html.
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Mamadatokhonova, Guldasta. "Detection of adenoviruses in cattle /." Uppsala : Dept. of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/10573858.pdf.
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Marttila, Marko. "Cellular receptors for species B adenoviruses." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1351.
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Hamdan, Salehhuddin. "Studies on the use of adenoviruses and adenovirus structural proteins in gene transfer to human cells." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414283.
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Alissa, Alkhalaf Moustafa. "Molecular analysis of adenoviruses from clinical samples." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/molecular-analysis-of-adenoviruses-from-clinical-samples(4b2e8cca-da89-4c8f-a4cc-8dffc489fa49).html.
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At present, 56 types of human adenovirus (HAdVs) have been identified and found to be associated with a variety of clinical features in the respiratory tract, eye, gastrointestinal tract, and other organs. In additions, HAdVs are able to establish persistent and latent infections in humans. Most of the work which has been carried out recently is related to adenovirus vectors and little has been done in other areas such as the nature and mechanisms of adenovirus persistence and latency in human tissues. Another area needing more investigation is the stability of the adenovirus genome which is useful for the development of adenovirus vectors and vaccines and for better understanding of adenovirus evolution especially with conflicting views about this issue.Recombination between two types of adenovirus can happen when the hexon epitope from one type and the fibre epitope from another type are found (intermediate strains). These recombinants can be detected by the conflicting results for serum neutralization (SN) and haemagglutination inhibition (HI) tests or by sequencing and phylogenetic analysis of the hexon and fibre regions of the adenovirus genome. The first part of this study is related to the stability and evolution of different adenovirus species. A total of 31 clinical isolates from AIDS patients previously typed in the hexon L2 region and the fibre knob region were analysed. These isolates were found to be from species D adenovirus (HAdV-D) and 28 of them had contradictory typing results in these two regions so they are clearly intermediate strains. Two isolates appear to be completely new and one isolate (Aids32) was typed as HAdV-D23 variant in both hexon L2 and fibre knob regions. Sequencing and phylogenetic analysis of the hexon L1, fibre shaft and penton regions of these adenoviruses revealed that no intragene recombination events occurred between the hexon L1 and L2 regions or between the fibre knob and fibre shaft regions. Sequencing and phylogenetic analysis of the penton showed that some of the intermediate strains had sequences from a third type of adenovirus in these regions. The penton analysis showed also that intragene recombination between penton HVR and RGD loop regions was common. New types of adenovirus were detected and sequential infection with different adenovirus variants was observed in some patients which indicates that the genome of HAdV-D from AIDS patients are not stable. Full genome sequencing and analysis was carried out for three isolates, two of them appeared to be new types of HAdV-D and the result of multi-recombination events and the third isolate appeared to be a variant of HAdV-D23.The stability of species B adenovirus (HAdV-B) was also analysed. A total of 96 isolates collected from the Manchester area typed previously by serum neutralization (SN) were analysed in five genome regions. Most of these isolates were HAdV-B3 and HAdV-B7 collected during a 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 collected in different years following the outbreak in addition to other adenovirus types isolated from different years. The phylogenetic analysis results of all the isolates in the structural regions: hexon L2, penton and fibre knob were found to be consistent and no mismatches (hexon from one type and fiber from another type) were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable in general. The last part of this study is investigating adenovirus persistence and latency in human tissues. Tonsils and adenoids (106 right and left tonsils and 10 adenoids) were obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty four (72.41%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load was not the same in the right and left tonsils in most of the cases and ranged from 280 to more than 2.6 x 106 copies/107 cells. Seventy eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C types were detected in 82% of the samples followed by species B (7.7%), HAdV-E4 (7.7%) and HAdV-F41 (2.56%). No DNA methylation was detected in the major late promoter (MLP) and E1A promoter regions of six tonsils and adenoids samples and two clinical isolates.
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Farrera, Sal Martí. "Enhanced hyaluronidase and tumor neoepitope expression by oncolytic adenoviruses." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671748.
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The oncolytic viruses (OVs) preferentially infect tumor and selectively replicate in cancer cells without harming normal tissues. OVs have been tested in clinical trials as monotherapy or combined with chemotherapy, radiotherapy, and immunotherapy. Nonetheless, the intratumoral spreading and the immune response hamper the treatment efficacy. In this thesis, these two challenges have been addressed in three separate chapters.
First, VCN-01, a hyaluronidase-expressing oncolytic adenovirus, was tested in a clinical trial in pancreatic cancer patients. We assessed the immune response triggered by VCN-01 as monotherapy or in combination with chemotherapy. We reported an early anti-viral immune response induction of IL-6, IL-10, IFNγ, IDO1, IP-10, and sLAG-3 in serum, independently of chemotherapy. We found a correlation between treatment toxicity and the IL-6 and IL-10. Furthermore, the triggered anti-viral immune response such as IFNγ, sLAG-3, and neutralizing antibodies anti-Ad5 was associated with better antitumor activity in patients.
The neoepitope vaccines have been tested in patients with limited clinical responses. We hypothesized that an oncolytic adenovirus (OAd) encoding for stroma.
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Kallioinen, Susanna. "Modification of the E1-pIX region of the adenovirus 5 genome for use in cancer gene therapy /." St Andrews, 2008. http://hdl.handle.net/10023/442.
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Ganly, Ian. "E1B attenuated adenoviruses in genetic therapy for cancer." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266588.
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Fajardo, Calderón Carlos Alberto. "Arming oncolytic adenoviruses with bi-specific T-cell engagers to improve antitumor efficacy." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/403492.
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Oncolytic adenoviruses that selectively replicate in cancer cells while sparing normal tissue have gained considerable attention as novel anticancer drugs. However, clinical trials with these viruses have identified the immune system as a major hurdle for their success in cancer patients. Despite the existence of a highly immunosuppressive tumor environment, adenovirus-infected cells can nonetheless be efficiently cleared by virus-specific infiltrating cytotoxic T lymphocytes without compromising tumor burden. We hypothesize that arming oncolytic adenoviruses with bi-specific T-cell engagers (BiTEs), a new class of antibodies that re-direct T-cells to cancer cells, might favor antitumor rather than anti-viral immune responses. We have engineered the oncolytic adenovirus ICOVIR-15K to express EGFR-targeting BiTEs under the control of the major late promoter. ICOVIR-15K armed with a BiTE targeting human CD3 and EGFR (ICOVIR-15K-cBiTE) was successfully rescued and it showed similar oncolytic properties as the parental virus. cBiTE expression and secretion was detected in supernatants from ICOVIR-15K-cBiTE-infected cells, and the secreted BiTEs bound specifically to both CD3+ and EGFR+ cells. In cell co-culture assays, ICOVIR-15K-cBiTE-mediated oncolysis resulted in robust T-cell activation, proliferation, and bystander cell-mediated cytotoxicity. Notably, intratumoral injection of this cBiTE-expressing adenovirus increased the persistence and accumulation of tumor-infiltrating T cells in vivo. Moreover, in two distinct tumor xenograft models, combined delivery of ICOVIR-15K-cBiTE with peripheral blood mononuclear cells or T cells enhanced the antitumor efficacy achieved by the parental counterpart. We also demonstrate that another oncolytic adenovirus expressing a chimeric BiTE targeting human EGFR and mouse CD3 (ICOVIR-15K-mcBiTE) induce robust mouse T-cell activation, proliferation, and cell-mediated cytotoxicity of cancer cells in vitro. Thus, ICOVIR-15K-mcBiTE is a promising surrogate of ICOVIR-15K-cBiTE that will aid in future pharmacological and toxicological preclinical studies of BiTE-armed oncolytic adenoviruses. Finally, we show that the combination of ICOVIR-15K-cBiTE with chimeric antigen receptor T-cell therapy can overcome some of the limitations encountered by both agents as monotherapies. The results described in this thesis demonstrate that BiTE-armed oncolytic adenoviruses hold properties with the potential of solving key limitations in oncolytic virotherapy, and encourage their further evaluation and development.Los virus oncolitos, capaces de infectar selectivamente células cancerosas sin afectar aquellas sanas, han despertado interés en los últimos años como nueva terapia contra el cáncer. Sin embargo, los ensayos clínicos con estos virus han demostrado que el sistema inmune supone un obstáculo para el éxito de los mismos en pacientes con cáncer. A pesar de la inmunosupresión que se observa en el ambiente tumoral, las células cancerosas infectadas por el adenovirus pueden ser eliminadas eficientemente por los linfocitos T anti-adenovirales sin comprometer la carga tumoral. La hipótesis de esta tesis es que adenovirus oncoliticos expresando bi-specific T-cell engagers (BiTEs por sus siglas en inglés) capaces de redirgirir los linfocitos T para atacar las células cancerosas, puede favorecer la respuesta inmune antitumoral sobre la antiviral. El genoma del adenovirus oncolitico ICOVIR-15K fue modificado genéticamente para expresar BiTEs contra el receptor del factor de crecimiento epidérmico (EGFR por sus siglas en inglés) bajo el control del promotor mayor tardío. El virus ICOVIR-15K expresando un BiTE que reconoce el EGFR y el CD3 humanos (ICOVIR-15K-cBiTE) fue generado y retuvo propiedades oncoliticas similares a la del virus parental in vitro. La expresión y secreción del cBiTE fue detectada en los sobrenadantes de células infectadas ICOVIR-15K-cBiTE, y sus propiedades de unión a células CD3+ o EGFR+ fueron confirmadas in vitro. En experimentos de cocultivos, la oncolisis generada por ICOVIR-15K-cBiTE indujo la activación y proliferación de los linfocitos T, y aumentó la citotoxicidad de células cancerosas. La inyección de este adenovirus aumentó la persistencia y la acumulación de linfocitos infiltrantes de tumor in vivo. Adicionalmente, experimentos en modelos murinos de cáncer basados en la administración combinada de ICOVIR-15K-cBiTE y linfocitos humanos demostraron un aumento en la eficacia antitumoral comparado con el virus parental. Por último, hemos demostrado que la combinación de ICOVIR-15K-cBiTE y linfocitos T con receptores de antígeno quiméricos (CAR por sus siglas en inglés) pueden superar muchas de las carencias que tienen ambas terapias. Los resultados de esta tesis demuestran que los adenovirus oncoliticos expresando BiTEs tienen propiedades que puede superar muchas de las limitaciones de la viroterapia del cáncer, y alienta a continuar su evaluación y desarrollo a nivel clínico.
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Brooks, Louise Alexandra. "Demonstration of new subtypes of adenovirus 7 in South Africa, and probing oesophageal carcinoma cell lines for evidence of adenovirus or of other oncogenic viruses." Master's thesis, University of Cape Town, 1987. http://hdl.handle.net/11427/25889.
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This study was carried out in 2 parts: 1. Genome analysis of human adenovirus species 7; 2. Search for a possible viral aetiology in oesophageal carcinoma. Sixteen laboratory isolates of adenovirus species 7, isolated in South Africa between 1975 and 1986, were characterized by restriction endonuclease analysis of their DNA genomes. Virus was propagated in human embryo fibroblast cells; genomic DNA, extracted and purified from cellular DNA extracts, was analyzed using 9 different restriction enzymes. Results of this study have demonstrated 2 new genome types of adenovirus 7c which have not previously been identified. The 2 novel strains, designated as genome types Ad7c1 and Ad7c2, were shown to differ from prototype Ad7 c according to restriction profiles with EcoRI; 2 new EcoRI sites were demonstrated in Ad7c1 and 1 in Ad7c2. The restriction sites were mapped on the viral genomes (at 3.68kb and 5.32kb from the left terminus) by double enzyme digestions, cloning of viral DNA, and nucleic acid hybridization using a cloned Ad7 probe. Strains resembling the prototype Ad7c and Ad7p (Gomen) genome types were also identified in the 1985 and 1986 Ad7 isolates. In order to investigate the possible role of a viral co-factor in the aetiology of oesophageal carcinoma, 18 probes, derived from potentially oncogenic viruses, were used to screen 3 human oesophageal carcinoma cell lines for the possible presence of integrated viral DNA. One of these, an Ad7 recombinant plasmid probe, was developed by cloning DNA from the transforming region of the Ad7cl strain into the plasmid vector pUC19. Cellular DNA, extracted from the 3 tumor lines HCU18, HCU33 and HCU39, was tested by means of both DNA dot hybridization and Southern blot hybridization for the presence of Epstein-Barr virus, human papillomavirus (types 1, 5, 6, 8, 11, 16, 18), human adenovirus (strains 5, 7, 12, 31) and human T-lymphotropic virus type I DNA. Both assays were demonstrated to be sensitive enough to detect 1 copy of viral DNA per cell. No hybridization between HPV, EBV, HTLV-I or adenovirus DNA probes, and the cellular DNA was detected. These findings indicate that the stable integration of these tumor viruses in host chromosomes did not play a role in the maintenance of the malignant phenotype of the 3 extensively passaged cell lines. Cells of the 3 oesophageal tumor lines were further examined by transmission electron microscopy, but the presence of virus particles in these cells was not observed.
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Corbin-Lickfett, Kara A. "Investigating the mechanisms used by the Adenovirus E4-34kDa protein to promote viral late gene expression." Oxford, Ohio : Miami University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1053305757.
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Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2003.Title from first page of PDF document. Document formatted into pages; contains v, 78 p. : ill. Includes bibliographical references (p. 68-78).
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Francini, Nora. "Polymer coating for the systemic delivery of oncolytic adenoviruses." Thesis, University of Nottingham, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.718458.
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Oncolytic adenoviruses have demonstrated great potential in terms of activity and safety when administered directly to confined solid tumors. However, the lack of routes to systemically deliverthese viruses safely and efficiently, restricts their clinical application, due to host immune response, undesired interactions with blood components and a hepatic tropism, thus decreasing their bioavailability at the tumor sites and so their efficacy. Efficacy of systemically administered viruses has been improved by masking viral surface proteins with polymeric materials, through modulation of viral pharmacokinetic profile and accumulation in tumors in vivo. Although some promising results have been achieved within this area, a clear understanding of the polymer features required to obtain the desired "stealthing" without compromising the activity of the virus is still lacking. In this thesis we sought to elucidate structure-activity relationship of N-(2-hydroxypropyl) methacrylamide (PHPMA) co-polymers and their ability to efficiently circumvent neutralization of ColoAd1, a chimeric oncolytic virus, as a mean to improve systemic virotherapy. Firstly, suitable synthetic strategies were developed that gave access to libraries of novel PHPMA functional copolymers which share identical macromolecular features but differ for the relative amount of reactive functionalities. Polymer coating covalently bound to viral surface proteins enhanced protection against neutralizing Page | ii Abstract antibodies in vitro. In vitro and in vivo viral infectivity was found to be inversely proportional to the coating efficacy. We demonstrated that a loss in viral infectivity resulting from polymer coating is not due to hampered cell entry and may not be a permanent inactivation of the virus. Preliminary observations suggest that virus activity may be restored by tailoring the polymer design, without compromising the virus "stealthing" effect.
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Storm, Rickard. "Early host cell interactions and antivirals against ocular adenoviruses." Doctoral thesis, Umeå universitet, Virologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-99907.
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Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC. EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber. Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC. After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells. Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.
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Martins, Sandra Soares. "Adenovirus e rotavirus como indicadores biologicos em aguas residuarias de esgotos sanitarios apos tratamento por processo anaerobio e disposição controlada no solo." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317116.
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Orientador: Maria Silvia Viccari GattiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-09T22:09:52Z (GMT). No. of bitstreams: 1 Martins_SandraSoares_M.pdf: 1140484 bytes, checksum: de8846c9246eef9258a4b0d3953a88b6 (MD5) Previous issue date: 2006
Resumo: O tratamento de esgoto sanitário em lagoa de decantação anaeróbia e disposição controlada de água residuária no solo é uma alternativa de baixo custo para o reuso de águas residuárias na agricultura. Nele, o esgoto sanitário é depositado em uma lagoa de decantação anaeróbia com retenção hidráulica de sete dias, após o que a água residuária é conduzida para rampa de solo franco argilo-arenoso, com cobertura vegetal de gramínia Cynodon sp, para disposição por escoamento superficial, seguindo-se sua infiltração e percolação. O objetivo desse trabalho foi verificar a eficiência desse sistema na eliminação e/ou inativação de adenovírus humanos (HAdV) e rotavírus (RV). Amostras de 1L de água residuária foram obtidas em quatro coletas, em intervalos de sete dias, na entrada do esgoto bruto (EB), no ponto de aplicação na rampa (0m) e nos pontos da sua superfície após 10, 20, 30, 35m e 40m. Sob a rampa, a 1m de profundidade e distantes 30m (30-1) e 35m (35 -1), dois pontos foram amostrados. Antes do ponto 0m, pontos de testemunha a 1m de profundidade e distantes 2m (T1) e 0,5m (T2) da rampa, e um ponto do lençol freático (LF) a 3m de profundidade, também foram coletados.As água residuárias foram concentradas de 1.000 a 5.000 vezes por filtração e eluição em membrana eletropositiva e ultracentrifugação. Nos eluatos obtidos, após extração de DNA, a presença de HAdV foi pesquisada por PCR e nested-PCR. Para a detecção de RV usou-se RT-PCR e duplo-semi- nested-PCR. Eluatos HAdV positivos foram inoculados em células HEp-2 e após até cinco passagens a presença de HAdV foi confirmada por PCR.. HAdV foram detectados em 29 das 35 amostras analisadas, sendo positivos todos os pontos de EB e da superfície da rampa. Em profundidade, sob a rampa, quatro amostras foram positivas, além de outras duas em T2 e uma em LF, o que demonstra a percolação desses vírus no solo com contaminação do LF. Quando testadas em células HEp-2, nas amostras do EB e dos pontos 0, 30, 35 e 40m a presença de vírions foi determinada, enquanto nos pontos 30-1m e LF os HAdV não foram infectivos. Esses resultados permitem concluir que o sistema não foi eficiente para remover e/ou inativar HAdV. Por outro lado, pode-se afirmar que os HAdV são indicadores virais adequados para esse sistema, desde que mantida a metodologia aqui empregada. Uma amostra de EB foi positiva para RV (genotipos G1 e G2), resultado esse que não permite qualquer conclusão. Para o reuso da água residuária advinda desse sistema impõe-se a associação de processos de desinfecção para a eliminação de HAdV
Abstract: Urban sewage treatment by an anaerobic process with overland flow system is a cheap alternative to reuse domestic effluents in agriculture. In this procedure, wastewater remains in an anaerobic pond for seven days, and then it is spilled from the top of a 40-meter extension slope covered with Tifton 85 (Cynodon sp) grass in order to surface flow and percolate until it reaches groundwater. The objective of this work is to determine if this procedure could be effective in removing and/or inactivating human adenoviruses (HAdV) and rotaviruses (RV) in a test unit in Limeira - SP, Brazil. Samples were collected every seven days from different spots in four sampling events totalizing one liter of wastewater. Sampling points were chosen at the raw sewage (EB), on the surface of the slope at 0, 10, 20, 30, 35 and 40 meters, and down one meter from the surface of the slope at the 30- and 35-meter points (30-1 and 35-1). Other points upslope were used at a distance of 2 meters (T1) and 0.5 meters (T2), beyond a 3-meter depth and 1-meter distant spot (LF). All samples were concentrated from a 1000 to 5000 times by filtration through electropositive microporous membrane followed by ultracentrifugation. HAdV detection was performed by both PCR and nested PCR. RV detection was accomplished by both RT-PCR and duplex semi-nested PCR. The positive samples for HAdV were inoculated in HEp-2 cells, and confirmation of the virions was performed by PCR. HAdV were detected in 29 of the 35 samples tested including in all samples both from the EB and from the surface of the slope. HAdV tested positive in the two T2, in one LF, and in the four samples underneath the slope. In HEp-2 cells HAdV virions were detected at the EB and at 0, 30, 35, and 40 meters on the surface of the slope. Spots 30-1 and LF were tested in HEp-2 cells, resulting negative to the presence of infective viral particles, although they tested HAdV positive. These results attest to the inefficiency of the proposed system of sewage treatment in removing and/or inactivating HAdV; however, maintaining the methodology used in this research, HAdV proves to be the appropriate viral indicator in this system. In relation to RV, no conclusions can be extracted since just one sample from the EB was RV-positive (G1 and G2 mixture). Finally, before reuse in agriculture, the effluents from the anaerobic pond should be disinfected to eliminate these viruses
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular
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De, Silva Shamika Udayangi. "Chimeric adenoviruses as potential gene therapy vectors for HIV vaccination." Thesis, Royal Holloway, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435928.
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Andersson, Emma. "Human adenoviruses : new bioassays for antiviral screening and CD46 interaction." Doctoral thesis, Umeå universitet, Virologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-35733.
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Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication. There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not. CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59. These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.
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Carr, Sharon. "Adenovirus and its interaction with host cell proteins /." St Andrews, 2007. http://hdl.handle.net/10023/219.
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Stevenson, Fiona B. "Preliminary characterisation of the adenovirus type 40 E1A region." Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323319.
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Wilkinson, D. S. "Studies of the Vertical Tranmission of Adenoviruses and Astroviruses in chickens." Thesis, Queen's University Belfast, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.527904.
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Sherwood, Victoria. "Human enteric adenoviruses and their interaction with the host interferon response." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431924.
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Adam, Virginie Sarah. "Prostate cancer targeting using replication-selective adenoviruses in combination with phytochemicals." Thesis, Queen Mary, University of London, 2009. http://qmro.qmul.ac.uk/xmlui/handle/123456789/204.
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Oncolytic adenoviral mutants have demonstrated good safety profiles but, despite some encouraging clinical results, efficacy as a single agent was limited. Combinations with conventional chemo- or radiotherapy significantly enhanced the anti-tumour effect. We investigated the possibility of enhancing prostate cancer (PCa) cell killing using adenovirus type 5 (Ad5) with phytochemicals. Phytochemicals are chemopreventive and can modulate intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway which regulates the expression of the coxsackie-adenovirus receptor (CAR). Equol and resveratrol synergistically enhanced cell death in both androgen receptor (AR)- positive and AR-negative PCa cell lines. On the other hand, curcumin, epigallocatechin-gallate (EGCG) and genistein had either synergistic and antagonistic responses with Ad5, depending on the dose and timing of addition. We therefore decided not to pursue the use of these compounds. Although we found that equol and resveratrol increased adenoviral receptor expression and viral uptake, this was not paralleled by enhanced viral replication. Treatment of DU145 and PC-3 cells with equol or resveratrol decreased viral titres, but did not block cell cycle progression. E1A expression in these cells was lower at 18h post-infection, but levels were normal by 72h. The exact mechanism behind the repression of adenovirus replication remains unclear. Equol and resveratrol induced moderate apoptotic responses. Mitochondrial membrane depolarization and caspase-3 activation were further increased by the addition of Ad5 in DU145 and PC-3 cells, but was reduced in 22Rv-1 cells. Caspase inhibition could not prevent sensitisation of 22Rv-1 cells to combination-induced cell death. The involvement of additional cell death mechanisms was therefore considered. Equol and resveratrol triggered autophagy while Ad5 acted as an autophagy repressor. Modulation of autophagy using pharmacological inducers and repressors indicates that autophagy may play a protective role in AR-negative cell lines.
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Davids, Michaela. "Molecular characterisation of human adenoviruses from environmental samples in Tshwane, Gauteng." Diss., University of Pretoria, 2019. http://hdl.handle.net/2263/78754.
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Human adenoviruses (HAdV) are non-enveloped viruses with an icosahedral capsid and a linear double-stranded DNA genome. These viruses belong to the family Adenoviridae and genus Mastadenovirus. An important property of the HAdV is that it is non-enveloped making it highly resistant to detergents and harsh environmental conditions. This virus is grouped in seven species (A-G) with more than 88 genotypes. These seven species are associated with several diseases, such as, respiratory infections, keratoconjunctivitis, urinary infections, hepatitis and gastrointestinal infections. The HAdV is one of the etiological causes of acute gastroenteritis, mainly caused by HAdV-F40 and HAdV-F41. The virus can be transmitted via the faecal-oral route, inhalation of respiratory droplets and direct contact with contaminated environments. The virus is known to be ubiquitous in environments where human contamination is likely to occur such as wastewater treatment plants. These human contaminations could occur through contaminated secretion and excretions within aqueous environments. There is currently a limited amount of information on the HAdV in water Molecular characterisation of human adenoviruses from environmental environments, particularly in Tshwane, Gauteng. Therefore, the aim of the study was to investigate the presence and genotypes of human adenovirus in environmental samples namely raw sewage and treated effluent, using molecular methods. For genotypic characterisation, Sanger sequencing was used on amplicons from 12 HAdV positive samples and next generation sequencing were used on all the amplicons from HAdV positive samples.
A total of 150 environmental samples (75 raw sewage and 75 effluent) were collected from two wastewater treatment plants in Tshwane over the study period of 18 months. These environmental samples comprised of 1 L raw sewage and 10 L treated effluent samples. The primary viral recovery for the 1 L raw sewage and 10 L treated effluent samples were performed using skimmed milk flocculation procedure and glass wool adsorption elution technique, respectively. For secondary viral recovery, both environmental samples were subjected to polyethylene glycol/sodium chloride precipitation. Manual extraction was used to extract the nucleic acids from the virus concentrate with mengovirus (MV) used as an extraction control. For the quantification of HAdV, standard curves prepared from known dilutions of HAdV and MV were used.
Human adenovirus was detected in 140/150 (93%) of the environmental samples comprising of 69/75 (92%) being raw sewage and 71/75 (95%) being effluent samples. The HAdV concentrations detected in wastewater treatment plant 1 (WWTP 1) ranged from 1.38x105 gc/L to 4.50 x 109 gc/L for raw sewage and 5.08x103 gc/l to 4.30x108 gc/L for effluent. The HAdV concentrations detected in WWTP 2 ranged from 6.84x104 gc/L to 1.69x1012 gc/L for raw sewage and 5.27x103 gc/L to 1.16x108 gc/L for effluent. The HAdV hexon amplification success rate from the nucleic acids was 43/140 (31%).
Eighteen HAdV genotypes were successfully characterised using Sanger sequencing. The HAdV-D was the most predominant species in both WWTPs, follow by HAdV-B and HAdV-F. The HAdV-A and HAdV-E species were the least identified. Next generation sequencing identified four times as many genotypes as Sanger sequencing (77 different genotypes). The HAdV-D (types 8, 9, 13, 17, 19, 20, 23, 24, 28, 29, 32, 33, 36, 42, 44, 47, 49, 51, 56, 60, 62, 64, 67 and 81) and HAdV-B (types 2, 3, 7, 11 and 66) were the most predominant species followed by HAdV-F (types 40 and 41), HAdV-A (types 12 and 76), HAdV-E ( type 4) and HAdV-C (type 1).
Testing wastewater treatment plants is advantageous as it allows for the detection and identification of HAdV types circulating in the surrounding communities. Due to the large number of species identified using NGS, it is the superior typing method and should be used for future studies. These include strains causing symptomatic and asymptomatic infections. Human adenovirus was detected at comparable frequencies in raw sewage and treated effluent wastewater, with slightly higher detection in effluent samples. However, the viability of these viruses is unknown and should be investigated in further studies. The detection of viruses in wastewater treatment plants are a public health concern as the treated effluent is discharged into rivers, which may be used by communities for domestic and recreational purposes.Dissertation (MSc (Medical Virology))--University of Pretoria, 2020.
NRF, PRF
Medical Virology
MSc (Medical Virology)
Restricted
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Chen, Yan, and 陳岩. "Recombinant adenovirus and adeno-associated virus mediated BMP2 and BMP4 gene therapy for new bone formation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31244038.
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Huang, Tiangui. "A study of adenovirus mediated transfer of p53 and Rb in cervical cancer cell lines." Click to view the E-thesis via HKUTO, 1999. http://sunzi.lib.hku.hk/hkuto/record/B42575114.
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Mirza, Momina. "Characterization of the cellular receptor for coxsackievirus and adenovirus /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-889-4/.
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Pearce, Oliver M. T. "Controlled virus glycosylation : engineering adenoviruses as targetable stealth vectors for gene therapy." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670156.
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Villanueva, Verdejo Eneko. "Nuevas estrategias de control postranscripcional en el desarrollo de adenovirus oncolíticos." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/397744.
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El cáncer de páncreas es la cuarta causa de muerte por cáncer en los países industrializados. En 2030, las predicciones señalan que puede pasar a ser la segunda. Pese a la relativa baja incidencia de este tipo de cáncer, su elevada mortalidad hace que la supervivencia a los 5 años no supere el 5%. El carácter sistémico de esta enfermedad, dado que en el momento del diagnóstico un alto porcentaje de casos presenta una patología localmente avanzada o con metástasis, hace que la cirugía sea una opción aplicable en un bajo número de pacientes. Además, esta neoplasia se caracteriza por presentar una elevada resistencia a quimio y radioterapia. Así, el desarrollo de nuevas terapias contra esta enfermedad resulta especialmente necesario.
Los recientes avances en las investigaciones oncológicas ponen de manifiesto el complejo entramado de desregulaciones de las células tumorales implicadas en el desarrollo del cáncer. Entre ellas, las alteraciones postranscripcionales han demostrado contribuir significativamente a la progresión tumoral, también, en el cáncer de páncreas. Este tipo de desregulaciones abren nuevas posibilidades para el diseño de terapias oncoselectivas basadas en agentes biológicos, como la viroterapia.
Así, en la presente tesis se han estudiado las implicaciones de las Proteínas de Unión a Elementos de Poliadenilación Citoplasmática (CPEBs), el uso de codones de las distintas proteínas adenovirales, y la incorporación de dianas para miRNAs desregulados en condiciones tumorales, como elementos de regulación postranscripcional capaces de conferir selectividad tumoral a la expresión de proteínas virales.
De esta manera, hemos demostramos que resulta posible explotar la reprogramación postranscripcional CPEB-dependiente con el fin de dotar de especificidad tumoral a la expresión de transgenes. Cuando el transgén controlado es el gen maestro adenoviral E-1A, responsable de la expresión del resto de genes virales, esta regulación confiere oncoselectividad a los adenovirus.
Así mismo, también demostramos que, a diferencia del resto de proteínas estructurales del virus, la fibra adenoviral presenta un uso de codones desoptimizado para su expresión en células humanas. Así, mientras que la optimización del uso de codones de la fibra mejora su expresión cuando ésta proteína se expresa de manera aislada, los adenovirus que expresan la fibra optimizada presentan una expresión atenuada de la propia fibra y del resto de proteínas estructurales del virus, generando una replicación viral deficitaria. En su conjunto, nuestros datos sugieren que el uso
balanceado de codones entre las proteínas de expresión tardía de los adenovirus favorecería su óptima traducción.
Por último, demostramos que la introducción de dianas para miRNAs desregulados en condiciones tumorales en el 3’UTR de genes estructurales, es una estrategia capaz de generar nuevos adenovirus cuya replicación se vea restringida a las células tumorales.Pancreatic cancer is he fourth cause of cancer-related death in developed countries. In 2030 it is expected to become the second one. Despite the relative low incidence of this cancer type, its high mortality leads to a survival after 6 months of 5%. The systematic character of this illness, associated to its high metastasis levels, makes surgery a suboptimal solution in the majority of cases. Besides that, this cancer type presents a high resistance to chemo and radiotherapy, making the development of alternative therapies a must. Recent advances in oncology research have highlighted the complex alteration of regulatory networks leading to cancer development. Among them, posttranscriptional modifications have proven to be necessary to allow tumour progression — also in the case of pancreatic cancer. However, this type of modifications is not easily controlled by conventional treatments. Conversely, they open new possibilities to therapies based on biologic agents, such as virotherapy. In this thesis, we have proven that posttranscriptional reprogramming can be exploited to generate tumour specificity in transgene expression. When the controlled gene is the adenoviral master gene El A, responsible for the expression of the rest of viral genes, this regulation confers tumour specificity to adenoviruses. Furthermore, we have also proven that there is a balanced codon usage in adenoviral proteins that favours their translation probably by allowing an optimal distribution of cellular translational resources. In the near future, this type of posttranslational modulation could allow the design of new adenoviruses in which therapeutic proteins could be expressed without compromising viral fitness. Finally, we demonstrate that the introduction of tumour deregulated miRNA targets in the 3'UTR of structural proteins is a new strategy to generate adenoviruses with an expression pattern restricted to tumour cells.
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Russell, Iain Alasdair, and n/a. "Involvement of p53 and Rad51 in adenovirus replication." University of Otago. Dunedin School of Medicine, 2007. http://adt.otago.ac.nz./public/adt-NZDU20070521.094929.
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As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery.
Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression.
Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle.
The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.
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Wiles, Karen Anna, and n/a. "Coxsackie and Adenovirus Receptor (CAR) expression is a potential limiting factor in adenoviral oncotheraphy." University of Otago. Dunedin School of Medicine, 2007. http://adt.otago.ac.nz./public/adt-NZDU20070619.161353.
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Novel approaches to cancer treatment in the context of Gene Therapy have been gaining popularity as an alternative to conventional therapies which have proven to lack specificity, resulting in tumour cell resistance, tumour progression and mortality. As a consequence the use of adenoviruses has been widely developed not only as a replication deficient vector for gene therapy but also as a replication competent oncolytic agent designed to selectively target and kill tumour cells. Unfortunately their success in clinical application has been limited, and it has been suggested that a lack of the primary viral attachment receptor 'CAR' could be a barrier to infection by limiting access to target cells. If Ad/CAR binding is the rate limiting step for successful Ad therapy, it is essential to establish a CAR expression profile in normal and tumour tissue, and in tumour progression, to enable more effective targeted therapy. Furthermore, in the context of using adenovirus as an anticancer strategy by exploiting its replicative lysis, it is important to explore whether Ad success is affected by CAR expression and to identify factors downstream of CAR that may be influential in this process.
In the first experimental chapter, an in vivo immunohistochemical analysis of tissue array slides determined CAR expression in a range of normal and tumour tissue. CAR was differentially expressed dependent on cell of origin, with normal stem cells and basal cells displaying very high CAR, signifying its importance in early development and differentiation. Epithelial cells were also high in CAR but its expression was negligible in mesenchymal, lymphoid and neural cells. This trend was also reflected in most tumour tissue albeit with a general decrease in CAR compared to corresponding normal tissue of the same organ. An exception was the blastic tumours which displayed high CAR reflecting their embryonic state of derivation. CAR expression also decreased in high grade, poorly differentiated tumours of the prostate, stomach and breast compared to their well differentiated counterparts.
In the second experimental chapter, a more comprehensive study of breast cancer biopsy specimens was undertaken, to determine both the expression of CAR and the tumour suppressor gene p53 in relation to tumour grade. The rationale being that both loss of CAR expression and p53 mutation (resulting in loss of function), have been associated with tumour progression. It is possible that CAR and p53 interact directly or indirectly and may be modulated by each other. This study revealed a decrease in both CAR and hormone receptor expression and an increase in p53 'mutational' status with increasing tumour grade. These three factors when compared independently to tumour grade are statistically significant associations, implying that CAR expression and hormone responsiveness decrease with tumour progression and p53 function is compromised or lost via mutation. There was also a significant association between CAR expression and hormone receptor status, however a significant association between CAR expression and p53 status within the tumour grades was not found.
Treatment outcome with Ads will also depend on defining factors downstream of CAR attachment that affect adenovirus 'permissivity', which is ultimately measured by viral replication and cell death, relying on the bystander effect to eradicate all tumour cells. The in vitro analysis revealed statistically significant associations between CAR receptor expression, 'infectivity' (virus infection) and permissivity. Cell lines that were more susceptible to Ad5 were generally of epithelial origin, had high CAR, and were easily infected. However there were exceptions and CAR was not the sole determinant in adenovirus cell entry nor in its ability to replicate and kill the cell. Permissivity was also related to p53 status. Thus, although CAR expression may indeed be a limiting factor, it is apparent that a combination of other events contributes to a deficient infection, especially in the deregulated tumour environment.
The results presented in this thesis clearly demonstrate that there is more to the story of 'CAR' which hints that its role in viro-oncotherapy is not limited solely to its function as an attachment receptor for adenovirus but may also involve its function as a cell adhesion molecule and signal transducer. The further elucidation of these aspects of CAR�s potential role in the scheme of tumour biology may alter the course and strategy of cancer therapy in the future.
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黃天貴 and Tiangui Huang. "A study of adenovirus mediated transfer of p53 and Rb in cervical cancer cell lines." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B42575114.
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Shen, Zan. "The kringle 1 domain of hepatocyte growth factor exerts both anti-angiogenic and anti-tumor cell effects on hepatocellular carcinoma." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40687661.
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Blixt, Ylva. "Early interaction between adenovirus type 2 and HeLa cells significance of the plasma membrane constitution /." Lund : Dept. of Microbiology, University of Lund, 1992. http://books.google.com/books?id=DzhrAAAAMAAJ.
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Sostoa, Pomés Jana de. "Oncolytic adenoviruses expressing transgenes targeting the tumor stroma to enhance the antitumor efficacy." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667027.
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Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor- associated stroma limits the efficacy of oncolytic viruses by forming a barrier that block efficient viral penetration and spread. Another important hurdle for the efficacy of OVs is the antiviral immune responses, where virus-specific infiltrating T cells clear adenovirus-infected cells without compromising tumor burden.
In this thesis, these hurdles have been addressed in separate chapters. We first hypothesized that arming an oncolytic adenovirus with a FAP-targeting bispecific T cell engager (FBiTE) could retarget infiltrated lymphocytes towards cancer-associated fibroblasts (CAFs), enhancing viral spread and favoring antitumor rather than anti-viral immune responses. The engineered ICO15K-expressing FBiTE virus showed similar infectivity and replication potency than the non-armed virus. FBiTE-mediated binding of CD3+ effector T cells and FAP+ target cells led to T-cell activation, proliferation, and cytotoxicity against FAP-positive cells in vitro. In vivo, FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. Finally, the antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus. The data presented in this thesis strongly supports that the combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP- expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development.
Aiming to induce stroma disruption, we secondly generated a panel of oncolytic adenoviruses expressing FAP-targeting immunotoxins and a nitroreductase (NfrA)- activatable prodrug. During the development of these projects, we successfully rescued and characterized all the viruses. However, although immunotoxins molecules were properly expressed and secreted from modified-virus infected cells, no promising results were obtained. In contrast, NfrA-armed virus showed replication-dependent enzymatic activity on target cells, leading to increased oncolytic potency in vitro. These preliminary results indicate that this last strategy could be considered to foster viral spread in stroma-abundant tumors, encouraging its validation in an in vivo setting.Les teràpies basades en virus oncolítics pel tractament de tumors sòlids es consideren molt prometedores degut a la seva capacitat de combinar la lisi directa de cèl·lules canceroses i la mort cel·lular per l’activació del sistema immune. No obstant, l’estroma associat al càncer forma una barrera que bloqueja la penetració i distribució del virus en el tumor, limitant l’eficàcia dels virus oncolítics. Una altra limitació important és la resposta immune contra el virus. Les cèl·lules T citotòxiques específiques contra el virus que infiltren el tumor eliminen, normalment, les cèl·lules infectades per l’adenovirus sense comprometre la massa tumoral. En aquesta tesi, aquestes limitacions han estat abordades en capítols separats. Primer vam hipotetitzar que un adenovirus oncolític armat amb un bispecific T cell engager (BiTE) contra FAP (FBiTE) podria redirigir els limfòcits infiltrats contra els fibroblasts associats al càncer (CAFs), millorant la distribució viral i afavorint la resposta antitumoral vers l’antiviral. El virus ICO15K que expressa el FBiTE va mostrar un patró d’infectivitat i de replicació similars al virus no armat. La unió de les cèl·lules T efectores CD3+ i les cèl·lules diana FAP+ mitjançada pel FBiTE va provocar l’activació, la proliferació i la citotoxicitat de les cèl·lules T contra la cèl·lules FAP positives in vitro. In vivo, l’expressió de FBiTE va induir l’acumulació intratumoral de les cèl·lules T i la disminució dels nivells de FAP, un marcador de CAFs, en els tumors. Finalment, l’activitat antitumoral dels adenovirus armats amb el FBiTE va ser superior que la del virus parental. Els resultats presentats en aquesta tesi aporten fortes evidències que la combinació de l’oncolisi viral de les cèl·lules canceroses i la citotoxicitat dels CAFs FAP+ mitjançada pel FBiTE pot ser una estratègia efectiva per superar les limitacions claus de la viroteràpia. Aquests resultats incentiven el desenvolupament d’aquesta estratègia pel seu ús en la clínica. Amb l’objectiu de destruir l’estroma, vam generar un panell d’adenovirus oncolítics que expressaven diferents immunotoxines específiques contra FAP i una nitroreducatasa (NfrA) activadora de prodroga. Durant el desenvolupament d’aquests projectes, vam obtenir i caracteritzar tots els virus. No obstant, encara que les diferents immunotoxines van ser adequadament expressades i secretades per les cèl·lules infectades pels virus, no vam obtenir cap resultat prometedor. El virus armat amb la NfrA, en canvi, va mostrar una activació enzimàtica depenent de la replicació del virus en les cèl·lules diana, incrementant la potència oncolítica del virus in vitro. Aquests resultats preliminars indiquen que aquesta última estratègia podria fomentar la distribució viral en tumors rics en estroma i incentiven la seva validació en models animals.
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Johansson, Susanne. "Design and Synthesis of Sialic Acid Conjugates as Inhibitors of EKC-causing Adenoviruses." Doctoral thesis, Umeå universitet, Kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1641.
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The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.
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Kostova, Youlia. "Armed YB-1 dependent oncolytic adenoviruses for combined virotherapy and suicide gene therapy." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-179119.
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Hall, Gregory John. "The in vitro effect of conditionally replication-competent adenoviruses in human astrocytoma cultures." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439587.
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Rohmer, Stanimira. "Novel strategies to improve the efficiency of therapeutic adenoviruses for the treatment of cancer." kostenfrei, 2010. http://d-nb.info/1002480280/34.
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Papp, Zsuzsanna. "Mucosal and systemic immune responses induced by immunisation of cotton rats with recombinant adenoviruses." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ27423.pdf.
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Dar, Nosheen. "Development of replication defective recombinant adenoviruses for the purpose of HIV-1 vaccine delivery." Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429507.
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Francis, Murray A. "Characterisation of DNA damage inducible responses and repair in human cells using recombinant adenovirus vectors /." *McMaster only, 2000.
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Chan, Yuk-on. "Impact of respiratory viruses on mortality." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/b39724025.
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Deol, Jatinderpal. "Development of helper-dependent adenovirus for gene expression in muscle." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33745.
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Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein on the cytoplasmic side of the sarcolemma. Gene therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD and other genetic diseases. These vectors are less immunogenic than their predecessors and have the capacity to carry large DNA inserts such as the full-length dystrophin (12 kb). However, the lack of viral genes results in a weakened and subsiding (short) transgene expression in muscle. Findings in the lung and liver have shown the adenoviral E4 region, in particular E4 open reading frame 3 (ORF3) to contribute to the maintenance of transgene expression. We constructed an adenovirus in which E4 ORF3 was reintroduced into a fully-deleted adenovirus along with full-length dystrophin (AdCBDysORF3). Dystrophin levels produced by AdCBDysORF3 were found to be not sustained in mdx mice, dropping significantly by day 90. However, expression levels did increase when AdCBDysORF3 was complemented with other viral proteins such as EIB. Likewise, increasing the expression of the primary adenovirus receptor (CAR) in muscle also resulted in a higher initial dystrophin expression in myofibers.
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Rodríguez, García Alba. "Enhancing the Antitumor Activity of Oncolytic Adenoviruses by Combining Tumor Targeting with Hyaluronidase Expression or by Increasing the Immunogenicity of Exogenous Epitopes." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/290068.
Full textAbstract:
Oncolytic adenoviruses represent an appealing therapeutic approach to treat cancer regarding its capability to infect and kill selectively tumor cells without damaging normal tissues. Tumor targeting upon intravenous administration and subsequent intratumoral virus dissemination are key features to improve oncolytic adenovirus therapy. To address these hurdles, in this work we have combined two different genetic modifications previously described by our group in an oncolytic adenovirus backbone with selective replication conditional to pRB pathway deregulation. First, the replacement of the heparan sulfate glycosaminoglycan-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK for tumor-targeting that has shown to prolong blood persistence and significantly enhance the therapeutic index compared with a nonRGDK-modified virus. Second, the expression of hyaluronidase to degrade the extracellular matrix and improve the intratumoral spread of the virus. Preclinical toxicology and biodistribution studies conducted in non-permissive mouse and semi-permissive Syrian hamster models supported the selectivity and safety of this novel virus, ICOVIR-17K. Antitumor efficacy was also demonstrated in different tumor models in immunodeficient mice and immunocompetent hamsters upon different routs of administration. Moreover, the combination of ICOVIR-17K with the chemotherapeutic drug gemcitabine further increased the antitumor activity of the virus. The data presented in this thesis strongly supports ICOVIR-17K as a promising clinical candidate which is currently being tested in phase I clinical trials.
Besides direct killing of cancer cells, oncolytic viruses may induce antitumor immune responses. Local inflammation of tumor tissue during an infection by an oncolytic virus provides suitable conditions to trigger antitumor immune responses against the tumor-associated antigens that are released in the immunogenic cell death process caused by these agents. Oncolytic adenoviruses can be used to promote immune responses against tumors by expressing and/or displaying tumor-associated antigens. However, a key limitation of this immunotherapeutic approach is the bias of the response towards the immunodominant viral antigens instead of the less immunogenic tumor antigens. In addition, defects in MHC class I antigen presentation pathway such as the downregulation of the transporter associated with antigen processing (TAP) are frequently associated with immune evasion of tumor cells and further impair the generation of specific immune responses against tumor antigens carried by oncolytic viruses. In this work we present a novel strategy that benefit from the TAP deficiency on tumor cells to enhance the response against those tumor epitopes, which had been attached to the viral protein E3-19K. This protein has a signal sequence that targets it to the endoplasmic reticulum, bypassing the necessity of TAP to transport the epitopes to that compartment. Compared to the display of the epitopes at the adenoviral capsid, this strategy promoted the immunogenicity of the epitopes and resulted in more potent antitumor immune responses, tackling a crucial aspect of virotherapy that is often overlooked.
In summary, the two different projects involved in this thesis addressed the main limitations of oncolytic adenoviruses from distinct points of view that may eventually be combined in order to maximize the opportunities of success of oncolytic adenoviruses in the clinics.La viroteràpia del càncer amb adenovirus oncolítics es basa en l’habilitat d’aquests agents en replicar selectivament en cèl·lules tumorals, produint la seva mort sense afectar cèl·lules normals. Les principals limitacions d’aquesta teràpia són la dificultat dels adenovirus per arribar als tumors després de ser administrats sistèmicament i també la seva incapacitat per dispersar-se de manera homogènia dins dels tumors. En aquest treball s’ha generat un adenovirus oncolític que combina dues mutacions descrites amb anterioritat pel nostre grup. Per una banda, la substitució del motiu d’unió a heparan-sulfats glicosaminoglicans situat al domini shaft de la fibra pel motiu d’unió a integrines RGD (mutació RGDK) per tal de millorar la ratio de transducció tumor/fetge i d’augmentar la persistència en sang de l’adenovirus. Per altra banda, l’expressió de hialuronidasa amb l’objectiu de degradar l’àcid hialurònic de la matriu extracel·lular del tumor i millorar la dispersió intratumoral de l’adenovirus. Aquest nou virus, l’ICOVIR-17K, va mostrar una potent eficàcia antitumoral en models de ratolí i hàmster que va ser fins i tot incrementada mitjançant la combinació amb gemcitabina, tot mantenint el perfil de toxicitat dels adenovirus oncolítics parentals. Per altra banda, a més de matar directament les cèl·lules tumorals, els adenovirus oncolítics poden contribuir a la generació de respostes immunes contra el tumor. El tipus de mort cel·lular que generen és altament immunogènic i ajuda al reclutament de cèl·lules del sistema immune que generen respostes contra els antígens tumorals alliberats en aquest procés. Una de les principals limitacions de la immunoteràpia amb virus oncolítics és la resposta esbiaixada cap als antígens virals, que són immunodominants, en lloc de cap als antígens tumorals, que són poc immunogènics. Per tal d’afavorir la generació de respostes immunes antitumorals, en aquest treball s’han incorporat epítops tumorals en la proteïna E3-19K de l’adenovirus, que conté una seqüència senyal que la dirigeix directament al reticle endoplasmàtic, de manera que evadeix els passos previs de processament antigènic per la via del MHC de classe I, comunament afectada en cèl·lules tumorals. Aquesta estratègia va permetre la generació de respostes immunes antitumorals més potents que quan els mateixos epítops eren incorporats a la càpside de l’adenovirus, i a més, van ser traduïdes en una millor eficàcia antitumoral en un model murí de melanoma. En resum, en aquest treball s’han abordat les principals limitacions dels adenovirus oncolítics des de diferents punts de vista que, eventualment, poden ser combinats per tal d’aconseguir un millor candidat per ser testat exitosament a la clínica.
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Addison, Christina Lynn. "Construction and characterization of adenoviral vectors expressing cytokines for cancer immunotherapy /." *McMaster only, 1997.
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Rovira, i. Rigau Maria. "Adenovirus oncoselectius pel tractament de càncer de pàncrees. Combinació d'estratègies de direccionament a tumor i bioselecció de microRNAs potenciadors de l'activitat adenoviral." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456987.
Full textAbstract:
L’adenocarcinoma ductal pancreàtic (PDAC) és una neoplàsia molt agressiva a conseqüència de la seva elevada capacitat metastàtica i la gran resistència que presenta als tractaments convencionals de quimioteràpia i radioteràpia. Només un percentatge molt reduït de pacients és elegible per a la resecció quirúrgica del tumor, l’únic tractament amb opcions de cura. La major part dels casos són diagnosticats en estadis avançats de la malaltia en els que la supervivència a 5-anys se situa al voltant del 7%. La millora del pronòstic dels pacients amb càncer de pàncrees ha estat pràcticament nul·la en els últims 30 anys, de manera que existeix una clara necessitat de desenvolupar nous mètodes per facilitar la detecció precoç del tumor i teràpies més efectives pel seu tractament.
Els adenovirus oncolítics estan esdevenint una teràpia prometedora pel tractament de neoplàsies agressives com el PDAC, obtenint resultats prometedors en assajos clínics. Tanmateix, els virus administrats fins al moment no han permès aconseguir respostes antitumorals complertes i és necessari disposar d’adenovirus més potents, però també de replicació selectiva en tumor.
En el primer bloc d’aquesta tesi, ens hem fixat en algunes de les desregulacions que presenten les cèl·lules tumorals per tal de dissenyar estratègies de direccionament a tumor i obtenir adenovirus oncoselectius més segurs. Concretament, l’increment de l’expressió de metal·loproteases de matriu, la reactivació en els tumors de vies relacionades amb el desenvolupament embrionari i la pèrdua de l’expressió de miRNAs específics de teixit han estat el racional per a generar modificacions genètiques que permetessin regular l’entrada dels adenovirus a la cèl·lula i l’expressió del gen E1A a nivell transcripcional i post-transcripcional. D’aquesta manera, s’ha determinat que la combinació de diversos mecanismes de control de la replicació viral permet obtenir adenovirus més segurs per una administració sistèmica tot mantenint una activitat antitumoral significativa.
En el segon bloc de la tesi, ens hem centrat en conferir més potència als adenovirus oncolítics. Vam hipotetitzar que les desregulacions en els perfils d’expressió de miRNAs de les cèl·lules tumorals podrien tenir un impacte negatiu en el cicle adenoviral, limitar la formació de nova progènia viral i per tant, reduir l’eficàcia antitumoral dels adenovirus oncolítics. Amb l’objectiu de contrarestar aquestes possibles limitacions i obtenir adenovirus amb una activitat antitumoral augmentada, es va dur a terme la bioselecció d’una biblioteca de miRNAs humans en adenovirus per tal d’identificar miRNAs potenciadors de l’activitat adenoviral en PDAC. A través d’aquest sistema high-throughput, es va determinar que el miR-99b i el miR-485 milloraven l’expressió dels gens virals i la formació de virions infectius en cèl·lules de PDAC gràcies a la regulació, directa o indirecta, de factors cel·lulars amb expressió diferencial en cèl·lules neoplàsiques i cèl·lules no tumorals. Així doncs, la identificació de miRNAs que milloraven el fitness viral va permetre obtenir adenovirus oncoselectius més potents front a PDAC.Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive neoplasia due to its high metastatic capacity and its resistance to chemotherapy. A small number of patients are eligible for tumor resection, the only curative treatment, and most of the cases are diagnosed at an advanced stage of the disease, in which the 5-year survival is around 7%. Therefore, there is a clear need for the development of better diagnostic methods and more effective treatments for this neoplasia. Oncolytic adenoviruses are becoming a promising therapy for the treatment of aggressive cancers, such as PDAC. Promising results have been obtained in clinical trials, although complete antitumoral responses have not been reached and more potent but also more selective viruses are required. In this thesis, we have focused in some deregulations present in cancer cells in order to design tumor targeting strategies for adenoviruses. Specifically, the overexpression of matrix metalloproteases, the reactivation of embryonic pathways and the loss of tissue specific miRNA’s expression have been the rational for the genetic modifications that allow the control of viral replication at transductional, transcriptional and post-transcriptional levels. We have determined that the combination of different strategies is useful for obtaining safer adenovirus for a systemic administration while maintaining a significant antitumoral activity. We have also focused in conferring more potency to oncolytic adenoviruses. We hypothesized that deregulations of miRNA profiles in cancer cells may have a negative impact on the adenoviral cycle, reducing the antitumoral efficiency of the virus. With the objective to counteract these limitations, we performed a bioselection of a human miRNA adenoviral library, aiming to identify miRNAs that confer potency to the adenoviruses against PDAC. We identified that miR-99b and miR-485 improved viral gene expression and the formation of infective particles in PDAC through the direct or indirect regulation of cellular factors differentially expressed in neoplastic cells and non-tumoral cells. Therefore, the identification of miRNAs that improved viral fitness gave rise to more potent oncoselective viruses against PDAC.
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Garcia, Moure Marc. "Generación de una adenovirus Ad5/52s pseudotipado con la proteína fiber corta del Ad52 para su caracterización in vitro e in vivo como vector de terapia génica." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/370116.
Full textAbstract:
La terapia génica consiste en la manipulación y utilización de material genético para el tratamiento de patologías. No obstante, esta estrategia requiere el uso de vectores de terapia génica para transportar el material genético al tejido diana de forma eficiente. Los vectores de terapia génica más comunes son los basados en el adenovirus humano de serotipo 5 (Ad5), porque, respecto a otros serotipos, el Ad5 tiene como ventaja su bioseguridad y facilidad de producción. Sin embargo, para que una célula sea transducida por el Ad5 ha de expresar un receptor reconocible por el Ad5, principalmente la proteína CAR, al que se une a través de su proteína fiber. El fiber de otros serotipos de adenovirus se une a receptores diferentes, permitiendo así la transducción de tipos celulares alternativos a los transducidos por el Ad5. De esta manera, una de las estrategias utilizadas para obtener un vector Ad5 con el tropismo de un serotipo diferente, es la pseudotipación del adenovirus, que consiste en substituir el fiber del Ad5 por el de otro serotipo. De entre los serotipos de adenovirus humanos, uno de los más recientemente descritos es el Ad52. Este serotipo contiene dos fiber de diferentes longitudes, lo cual es un rasgo compartido con los adenovirus humanos intestinales de la especie F. Como el Ad52 fue identificado a partir de muestras de heces de pacientes con gastroenteritis, se le atribuye también un tropismo intestinal, aunque no se ha podido demostrar. En el caso de los adenovirus de la especie F, su fiber largo reconoce el receptor CAR y, por lo tanto, se asume que su tropismo intestinal es mediado por el fiber corto. Dada la cercanía evolutiva entre el Ad52 y los adenovirus de la especie F, se asume que su fiber largo también reconocerá el receptor CAR, mientras que el fiber corto lo hará a otro receptor, dándole así al virus un tropismo independiente de CAR. Así pues, en esta tesis se ha generado un vector quimérico Ad5 pseudotipado con el fiber corto del Ad52 (Ad5/52s) con la finalidad de estudiar el rol específico de esta proteína fiber y, a la vez, caracterizar este adenovirus como vector de terapia génica. La primera parte de la tesis consiste en la generación del genoma del adenovirus Ad5/52s y la producción del vector, lo que incluye un estudio de su ciclo viral para optimizar la producción. El resultado del ciclo viral indica que éste se encuentra moderadamente retrasado respecto al del Ad5. Una vez producidos los vectores, se comparó su tropismo con el del Ad5 en cultivos in vitro, demostrando que la substitución del fiber comporta un cambio en el tropismo del Ad5 hacia líneas celulares, así como también aumenta su transducción en células de Schwann primarias, aunque no aumenta el tropismo del Ad5 en modelos intestinales in vitro. Ya caracterizado el virus in vitro, se demostró que el fiber corto del Ad52 no tiene afinidad por el receptor CAR, y su unión al receptor es dependiente del dominio knob del fiber. Posteriormente, el estudio comparativo de su distribución in vivo por administración intravenosa respecto al Ad5, mostró que el Ad5/52s tan solo transduce ligeramente pulmón a pesar de no ser secuestrado en hígado. Una vez descartados problemas de estabilidad, se observó que la baja eficiencia de infección del Ad5/52s se debe a una mayor inactivación de este virus en sangre, probablemente por la interacción con algún factor plasmático como la trombina. Finalmente, se modificó el fiber corto para limitar esta interacción, lo que permitió aumentar la supervivencia del Ad5/52s en sangre.Gene therapy is a biomedical approach, which consists of manipulating and delivering genes to treat a wide range of diseases. Nevertheless, a successful treatment also requires a vector to carry the therapeutic gene towards the targeted tissue and thus to increase itstransduction efficiency. Human adenovirus 5 derived vectors (Ad5) are the most commonly usedin gene therapy strategies due totheir higher biosafety and productivity. However, the Ad5 mediated transduction is restricted to those cells expressing appropriate viral receptors (mainly the CAR protein), recognized by the fiber protein. The fiber of other adenovirus serotypes binds to different receptors, allowing transduction of alternative cell types of those transduced by Ad5. Thus, one strategy to modify the natural Ad5 tropism is adenovirus pseudotyping, which consists of Ad5 fiber replacement by the fiber protein of another serotype. Among the different human adenovirus serotypes, one of the most recently described is Ad52. This serotype contains two fiber of different lengths, which is a common trait shared with enteric Ad40 and Ad41 human adenovirus. The Ad52 was initially found in stool samples obtained from patients with gastroenteritis and, as a consequence, it is supposed to be also an enteric virus, although it has not been demonstrated yet. In the specie F adenoviruses the long fiber recognizes the CAR receptor, and therefore, their enteric tropism is assumed to be mediated by the short fiber. Given the evolutionary relationship between the Ad52 and the species F adenoviruses, it is assumed that its long fiber also recognizes the CAR receptor, while the short fiber will recognize a different receptor, thus giving the virus a CAR-independent tropism. So, in this thesis we have generated a chimeric Ad5 vector pseudotyped with the Ad52 short fiber (Ad5/52s) protein, in order to study the specific role of the fiber protein, in turn, characterize this adenovirus as a vector for gene therapy. The first part of the thesis consists in the generation of adenovirus Ad5/52s genome and its production, which includes a study of its viral cycle to optimize production. The results show that the Ad5/52s viral cycle is moderately delayed compared to Ad5. Once the vectors were produced, the tropism of Ad5/52s and Ad5 were compared in in vitro, demonstrating that fiber replacement induces a switch in the Ad5 tropism in different cell lines, as well as an increased transduction in primary Schwann cells, but not in intestinal models in vitro. Then, it was also demonstrated that short fiber from Ad52 doesn’t recognizes the CAR receptor, and also that its binding is mediated by the fiber knob domain. Further studies to compare Ad5 versus Ad5/52 in vivo biodistribution after intravenous administration showed only a slight transduction in lung with Ad5/52s, despite not being trapped in liver. Once discarded stability problems, it was observed that the low levels of transduction achieved by the Ad5/52s were caused by a faster inactivation in plasma, probably by an interaction with a plasmatic factor, such as thrombin. Finally, to minimize this interaction, the short fiber was mutated, which enhanced the Ad5/52s survival in blood.
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Pauly, Maude [Verfasser], and Gerd [Akademischer Betreuer] Sutter. "Adenoviruses in Côte d`Ivoire: investigation of diversity and interspecies transmission / Maude Pauly. Betreuer: Gerd Sutter." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1076980902/34.
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Khan, Obaid Yusuf. "Construction of recombinant adenoviruses encoding skeletal troponin C protein and expression analyses in transduced cardiac myocytes." Thesis, University of Glasgow, 1998. http://theses.gla.ac.uk/5438/.
Full textAbstract:
Troponin C is a regulatory protein of the myofilament which binds to calcium to trigger the process of contraction. This protein exists in two isoforms, skeletal and cardiac, which are spatially and temporally regulated. Work in this project builds the primary stage of a long-term project, for using the gene transfer method to overexpress the skeletal isoform of Troponin C in cardiomyocytes. The long-term aim is to achieve complete or partial substitution of the native cardiac isoform and study the effects on contractile force produced, in normal and ischemic cardiomyocytes, both in vitro and in vivo. This project has involved designing, constructing and analyzing expression of adenoviral gene transfer vectors overexpressing the sTnC isoform. Several adenoviral vectors were generated with the wild type sTnC gene under the control of muscle-specific promoters. To facilitate analysis of protein expression and its subcellular localization, the sTnC protein was tagged with epitope tags and adenovirus generated, with this gene under the control of constitutive (CMV) and cardiac-specific (HCA) promoters. Epitope-tagged adenoviruses were expressed in vitro using mouse fibroblast (NIH3T3) cells and analyzed by western blot analysis, showing successful constitutive expression. Recombinant adenoviruses containing epitope-tagged-sTnC under the control of the human cardiac actin promoter showed cardiac-specific expression in cultured cardiomyocytes, in situ, using immunocytochemistry. The constitutively-expressing sTnC adenoviral vector showed successful expression in cardiomyocytes in culture, using northern blot analysis. A range of adenoviral vectors have been successfully generated, and constitutive and tissue-specific expression has been established for some of these vectors. Successes attained in this project have established the initial requirements to achieve the long-term goal to alter calcium sensitivity of myofilaments, by overexpression of sTnC isoform in cardiomyocytes, both in vitro and in vivo.
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Rojas, Expósito Luis Alfonso. "Blood barriers for oncolytic adenovirus efficacy: study of binding to erythrocytes via CAR and albumin‐mediated evasion of neutralizing antibodies." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/404054.
Full textAbstract:
Cancer virotherapy with oncolytic adenoviruses represents a promising therapeutic approach due to the capacity of these viruses to infect and selectively kill tumor cells without damaging normal tissues. Although the intravenous is the preferred route of administration in order to reach disseminated metastasis, several interactions with blood components cause the neutralization of the virus. Thus, improving the delivery of such adenoviruses to tumors by systemic injection is crucial for the success of the therapy. In this work we have studied the interaction of the adenovirus serotype 5 (Ad5) with human erythrocytes through the receptor CAR, which was described to sequester and inactivate the virus. Although erythrocyte binding was observed, it did not reduce viral transduction of tumor cells in vitro. Since mouse erythrocytes do not express CAR, human erythrocytes were transferred into nude mice to analyze the impact of erythrocyte binding after systemic administration. However, adenovirus extravasation and transduction of liver and tumors was not reduced, suggesting that this binding is reversible and does not neutralize the virus.
On the other hand, the high prevalence of anti-Ad5 neutralizing antibodies (NAbs) is a major obstacle for the intravenous administration of adenoviruses. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin, which allow them to maintain the infectivity and replication capacity in presence of NAbs. Non-modified adenoviruses are completely neutralized after systemic administration in pre-immune mice, whereas ABD-modified viruses preserve the ability to transduce target organs and induce oncolysis. The data presented in this thesis supports the use of this strategy to treat patients systemically with oncolytic adenoviruses.
In summary, this thesis focused on improving the intravenous delivery of oncolytic adenoviruses, which is one of the main limitations of this therapy. The results presented in this work demonstrate that while erythrocyte binding via CAR does not inactivate the virus, NAbs represent a major obstacle for efficacy. In this regard, albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus by systemic injection not only for cancer virotherapy, but also for gene therapy and vaccination.Els adenovirus oncolítics són agents terapèutics prometedors, degut a la seva capacitat d’infectar i eliminar selectivament les cèl·lules tumorals, sense afectar les cèl·lules normals. Tot i que la ruta preferida d’administració és la intravenosa per tal d’arribar a totes les metàstasis, la interacció del virus amb diversos components de la sang provoca la seva neutralització. Per tant, millorar l’arribada dels virus als tumors per via sistèmica és un aspecte clau per a l’èxit d’aquesta teràpia. En aquest treball s’ha estudiat la interacció de l’adenovirus serotip 5 amb els eritròcits humans a través del receptor CAR, la qual es va descriure que provocava el segrest i la inactivació del virus. Malgrat es va observar unió als eritròcits, aquesta no va reduir la transducció de cèl·lules tumorals in vitro. Degut a que els eritròcits murins no expressen CAR, es van transferir eritròcits humans a ratolins immunodeprimits per tal d’analitzar l’efecte de la interacció després de la injecció sistèmica. Tot i així, aquesta unió als eritròcits no va alterar la extravasació ni la transducció del fetge per part del virus, suggerint que la interacció és reversible i no neutralitzant. Per altra banda, l’alta prevalença d’anticossos neutralitzants contra l’adenovirus 5 en la població humana representa un obstacle molt important per la injecció intravenosa d’aquest. Per protegir l’adenovirus contra els anticossos neutralitzants s’ha inserit un domini d’unió a albúmina (ABD) a la proteïna principal de la càpside viral, la proteïna hexó. Aquest domini s’uneix a l’albúmina sèrica, recobrint el virus amb aquesta després de l’administració sistèmica. Els virus modificats amb ABD són capaços d’unir-se tant a l’albúmina humana com a la murina, fet que els permet mantenir la infectivitat i la capacitat replicativa en presència d’anticossos neutralitzants. Els adenovirus no modificats són completament neutralitzats després de la administració sistèmica en ratolins pre-immunes, mentre que els virus modificats amb ABD mantenen la capacitat de transduïr els òrgans i controlar el creixement tumoral. Els resultats presentats en aquesta tesi recolzen l’ús d’aquesta estratègia per a tractar pacients amb adenovirus oncolítics per via sistèmica.