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Chan, Kenneth See Kit. "Nef from pathogenic simian immunodeficiency virus attenuates vaccinia virus /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2004. http://uclibs.org/PID/11984.
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Wallengren, Kristina. "Envelopment of retrovirus and vaccinia virus /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4851-8/.
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Kettle, Susan. "Characterisation of vaccinia virus gene B13R." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294390.
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Moore, Jeffrey B. "Vaccinia virus 3[beta]-hydroxysteroid dehydrogenase." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359450.
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Keller, Brian Andrew. "Functional Genomic Studies of Vaccinia Virus Provide Fundamental Insights into Virus-Host Interactions." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36614.
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The oncolytic virus field is in the midst of strong and sustained growth. The clinical utility of this class of therapeutics has been bolstered in recent years by the rise of immune checkpoint inhibition, which has the potential to work synergistically with oncolytic viruses to increase the scope of patients who respond favourably to therapy. This growth has been further driven by clear industry support with several pharmaceutical companies acquiring or developing oncolytic virus products following the 2015 FDA approval of Talimogene laherparepvec and the generally-accepted potential of immunotherapeutic approaches to cancer treatment. Vaccinia virus is a double-stranded DNA virus with an extensive history of vaccine use in humans and a desirable safety profile. It is a large virus with a complex lifecycle, and its history of use as a vaccine has resulted in the generation of dozens of unique strains. Although it has been studied extensively, much remains unknown about many vaccinia virus gene function(s) and the virus’ interactions with cellular hosts. Vaccinia virus-based oncolytic viruses have been developed, however clinical outcomes thus far have been unsatisfactory. A more complete understanding of vaccinia virus gene functions must therefore precede the effective design of a next-generation vaccinia virus-based oncolytic candidate. With this downstream goal, we sought to (1) understand the unique oncolytic virus-relevant phenotypic properties of five clinical candidate vaccinia virus strains, and (2) generate and characterize a library of single-gene mutants of the Copenhagen strain of vaccinia virus. These studies resulted in the selection of vaccinia virus-Copenhagen as the wild-type strain of choice that will be utilized for future oncolytic virus development. Furthermore, the generation and initial characterization of an 89-member clonal library of vaccinia-Copenhagen single-gene mutants will be an important tool as we seek to generate a next-generation oncolytic virus candidate. Completed characterization studies challenge the role that viral thymidine kinase should play in oncolytic virus design, demonstrate novel functions of the vaccinia virus gene A47L, and provide an understanding of the role of the vaccinia virus gene F15L. These studies also raise the concept of the personalized selection of oncolytic virotherapeutics. This virus library has the potential to increase the fundamental understanding of vaccinia virus biology in this field as well as in the study of vaccine development and pathogen-host interactions.
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Bleckwenn, Nicole Aleece. "Protein production development with recombinant vaccinia virus." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1416.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2004.Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Gardner, Jeremy Damien. "Characterisation of the vaccinia virus gene A39R." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365448.
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Major, James R. "Interactions of dendritic cells with vaccinia virus." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401096.
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Odell, Mark. "An analysis of vaccinia virus DNA ligase." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670258.
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Napoli, Andrea. "Glycerophospholipid fluorescence imaging during vaccinia virus replication." Thesis, Sorbonne Paris Cité, 2019. https://theses.md.univ-paris-diderot.fr/NAPOLI_Andrea_1_va_20190415.pdf.
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Le virus de la vaccine (VACV) est l'organisme modèle pour l'étude des Poxviridae. Son cycle de réplication dans le cytoplasme de la cellule hôte a été largement étudié par microscopie optique et microscopie électronique. Grâce à des études génétiques approfondies, le rôle de certaines des 250 protéines du virus a été élucidé. Cependant, les mécanismes d’acquisition de la membrane du virus, notamment le rôle des lipides cellulaires impliqués, restent mal connus. L’étude de la composition des membranes de VACV purifiés par spectrométrie de masse a montré qu’elles présentent un enrichissement en acide phosphatidique (PA) et en dérivés de phosphatidylinositoles (PIPs). De plus, des études in vitro ont permis d’identifier certaines protéines virales capables de se lier aux PIPs in vitro. Le rôle de ces lipides dans le cycle de vie du virus, en particulier, dans la biogenèse de ses membranes n'a pas été identifié. L'objectif de ce projet de thèse est de déterminer l’implication du PA et des PIPs dans la biogenèse des membranes virales. L’expression transitoire de protéines recombinantes contenant des domaines de liaison à ces lipides a permis de déterminer la localisation du PA et des PIPs au cours de la réplication du virus. Afin de compléter ces résultats nous avons également utilisé des anticorps reconnaissant la PI4K et le PI4P. Enfin, l’utilisation d’inhibiteurs des PI3Ks et des PI4Ks a permis d’étudier le rôle de ces kinases durant l’assemblage de la membrane virale. A l'aide de ces outils, j'ai pu montrer que la localisation de ces lipides, à l'exception du PI3P, n'est pas altérée dans les cellules infectées. De plus, aucune co-localisation n’a été observée entre ces lipides et les sites de réplication du virus. Par ailleurs, nous avons observé une co-localisation entre le PI4P et les virus enveloppés ce qui est en accord avec les études précédentes montrant que les membranes du virus mature seraient dérivées de l'appareil de Golgi. Toutefois, des inhibiteurs de la synthèse du PI3P et du PI4P n'ont pas montré d’effets sur la production des membranes virales observables par microscopie optique. En conclusion, ce travail a permis de mieux définir le rôle des lipides durant la réplication de VACV. Ces résultats mettent en lumière un rôle potentiel du PI4P au cours de l’acquisition de l’enveloppe du virus ainsi qu’un rôle PI3P et de protéines reconnaissant spécifiquement le PI3P au cours des phases tardives de la réplicationVaccinia Virus (VACV) is the model organism for the study of the Poxviridae. Its cytoplasmic life cycle has been studied extensively by light- and electron microscopy. Thanks to a robust genetic system the role of some of its 250 proteins is beginning to be understood. Nevertheless, the acquisition of its membranes is still a matter of debate, in particular the role of cellular lipids. Lipid mass spectrometry of purified VACV previously showed an enrichment of phosphatidic acid (PA) and phosphatidylinositol derivatives (PIPs) in the viral membrane. Although some viral proteins have been shown to bind PIPs in vitro the role of these lipids in the viral life cycle, in particular viral membrane biogenesis, remains elusive.The aim of this work is to determine whether PA and PIPs are relocated in infected cells to the site of viral membrane biogenesis. For both PA and PIPs, I used recombinant proteins containing PA or PIP binding domains fused to eGFP, expressed them by transient transfection to follow their localization during viral replication. In addition, I used antibodies for the recognition of PI4K and PI4P. In order to understand the biochemical role of PIPs, I used pan-PI3K and PI4K inhibitors to study their effect on viral assembly. Using these tools, I could show that the lipids under investigation did not display an altered localization, with the exception of PI3P which showed a different pattern in infected cells. None of the PIPs analyzed co-localized with the sites of primary VACV membrane biogenesis. Consistent with the fact that the mature virus acquires additional membranes derived from the Golgi complex, I could show a co-localization of wrapped virus with PI4P, known to localize to this cellular organelle. However, drugs inhibiting PI3P and PI4P biosynthesis did not show any effect on VACV membrane biogenesis, at least at the light microscopy level. In conclusion, this work sharper defines the role of lipids during VACV replication. In particular, it opens the way to further studies on the putative role of PI4P during wrapping and the fate of PI3P and PI3P binding proteins during late replication
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Jarmin, Susan Anne. "Vaccinia virus protein A40 is an immunomodulator." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/4717.
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Vaccinia virus (VACV) strain Western Reserve gene A40R encodes a type II membrane glycoprotein with a C-type lectin-like domain at the C terminus. The A40 protein is not incorporated into virions, is nonessential for virus replication in cell culture and does not affect virus virulence in a murine intranasal model of infection. However, A40 does affect the outcome of infection in an intradermal infection model in which the virus lacking gene A40R produced smaller lesions and alterations in the host immune response. A40 has amino acid similarity to C-type lectins, such as NKG2A and DC-SIGN. This observation together with its location on the infected cell surface and its ability to bind to the surface of cells of the immune system is consistent with A40 functioning as an immunomodulator. It is possible that A40 might function by mimicking native host lectins or by modulating recognition of VACV-infected cells by cells of the immune system. To investigate the mechanism by which A40 affects the outcome of infection in vivo, a cloning and experimental strategy was devised to search for its ligand(s) and try to determine its structure in collaboration with protein crystallographers. To achieve these goals, the recombinant A40 protein has been produced in E. coli, mammalian cells, and from insect cells infected with recombinant baculoviruses. Bacterially expressed recombinant A40 was used to generate an antibody specific to A40 and this was then purified, characterised and used to further the characterisation of A40. The potential for A40 to interact with a ligand on the surface of another cell was investigated by a cell binding assay using recombinant A40 protein. This protein was produced in a mammalian system and was found to bind to the surface of immune cells but not to epithelial cells. In cytotoxicity assays, the absence or over-expression of A40 was found to modulate the ability of NK cells to kill VACV infected cells.
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Junior, Anselmo Vasconcelos Rivetti. "Patogenia do Vaccinia Virus GP2 em bovinos." Universidade Federal de Minas Gerais, 2012. http://hdl.handle.net/1843/BUOS-95ZG22.
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Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and milkers, and causing economical, animal and human health impacts. By the clinical presentation of the disease, it seems that BV is a localized disease, with lesions restricted to the
skin of affected individuals. But there are no studies about the pathogenesis of the disease in cows to access if there is a systemic spread of the virus and if there are different ways of VACV shedding. This work had the objective to study if occurs viremia and VACV shedding in the feces of VACV experimentally infected cows. To this end, ten crossbred lactating cows, serologically negative for VACV, were used. Three teats of each cow were scarified using sandpaper, followed by inoculation of 106 UFP/50µL of Guarani P2 (GP2V) strain of VACV.
All animals were monitored daily and blood and feces samples were collected for 67 days post infection (d.p.i.). After this period, all animals that were previously infected were divided in two treatment groups: re-infection or immunodeppression. All animals developed lesions
compatible with VB (papules, vesicles and ulcers), and even after the resolution of the lesions, viral DNA was detected in the blood and lymphoid tissues, as well as an intermittent and extended detection of VACV DNA in the feces, until the last day of collection (67 day post
infection), suggesting that VACV infection is chronic. The detection of VACV viable particles in the feces suggests that this is a possible route of viral shedding in the environment, which may favor VACV transmission within and among properties. VACV infections in cattle are associated with infection of epithelial cells and goblet cells in the intestine, and macrophages and lymphocytes in lymphoid tissues. In the animals experimentally infected and then immunodepression, VACV DNAnemia and DNA detection in feces were observed even before the immunodepressive treatment. There was a tendency to an increase in DNA detection in the blood and feces after the treatment, suggesting that there is some mechanism of VACV persistent infection in cattle and that this is influenced by the immune system. The group of animals that were reinfected by VACV, presented lesions in the teats once more, and VACV
DNAnemia was observed. This study showed new evidence that VACV infection in cattle is systemic, has a chronic course and that there is viral shedding on the feces.A vaccínia bovina (VB) é uma zoonose causada pelo Vaccinia virus (VACV), que afeta vacas leiteiras e seus ordenhadores, causando impactos econômicos e na saúde pública e animal. A apresentação clínica da doença é caracterizada pela presença de lesões localizadas na pele dos indivíduos afetados. Em bovinos não existem estudos sobre a patogênese da doença que descrevam a forma de disseminação do vírus, bem como suas vias de excreção. Este trabalho teve por objetivo estudar a ocorrência de viremia e da excreção do VACV nas fezes de vacas experimentalmente infectadas com este vírus. Para tanto, dez vacas mestiças, em lactação, sorologicamente negativas para o VACV foram utilizadas. Três tetos de cada vaca foram escarificados utilizando lixa e inoculadas com 106 UFP/50µL de VACV, amostra Guarani P2. Os animais foram acompanhados e coletadas amostras de sangue e fezes durante um período de 67 dias pós-infecção (d.p.i.), quando foram então divididos em dois grupos de tratamento, sendo um submetido à reinfecção e o outro à imunodepressão. Todos os animais desenvolveram lesões compatíveis com a VB (pápulas, vesículas e úlceras). Mesmo após a resolução das lesões, o DNA viral foi detectado no sangue e em tecidos linfóides e de forma intermitente e prolongada nas fezes dos animais até o último dia de coleta (67º dia pós-infecção), demonstrando que a infecção causada pelo VACV é prolongada. A detecção de partículas virais infecciosas nas fezes sugere que essa via de excreção seja uma forma de disseminação do vírus no ambiente, podendo favorecer a transmissão do VACV dentro e entre propriedades. Infecções do VACV em bovinos estão associadas à infecção de células epiteliais e caliciformes no intestino, e em macrófagos e linfócitos em tecidos linfóides. Os animais infectados e posteriormente imunodeprimidos experimentalmente, voltaram a apresentar DNAnemia e permaneceram eliminando o VACV nas fezes com uma tendência a um aumento nesta excreção, podendo sugerir que exista algum mecanismo de persitência na infecção do VACV em bovinos e que este seja influenciado pelo sistema imunológico. Os animais reinfectados pelo VACV voltaram a apresentar lesões nos tetos e DNAnemia. Este estudo demonstra novas evidências de que a infecção do VACV em bovinos seja sistêmica e prolongada, e que ocorra excreção viral nas fezes.
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Rehfeld, Izabelle Silva. "Vaccinia bovina em vacas secas e lactantes experimentalmente inoculadas com o Vaccina virus." Universidade Federal de Minas Gerais, 2011. http://hdl.handle.net/1843/BUBD-9D7J9H.
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This dissertation was divided into three experiments whose aims were to reproduce the bovine vaccínia (BV) in crossbred dairy and dry cows through the experimental inoculation with Vaccinia virus Guarani P2 (VACV-GP2). Experiment 1 studied the clinical and pathological evolution of the lesions and the localization of the virus by immuno-histochemistry in different stages of the disease. In this experiment, the teats of six crossbred dry cows, divided into three groups, were scarified with a hypodermic needle in the central area of each teat. Two groups were euthanized and necropsied in different stages of the disease, while in a third group, a biopsy was performed in the inoculated area of the teats In the experiment 2, three crossbred cows were inoculated in order to analyze different scarification methods and the pathogenicity of two different inocula with VACV-GP2. One cow was euthanized on the 17th day post-inoculation (d.p.i), period in which the lesions were healed. The aims of experiment 3 were to carry out clinical, hematological and biochemical analysis associated to the infection and to observe the effects of immunodepression and re-infection with VACV-GP2 in dairy cows previously infected by VACV. The experiment was divided into two parts. In part 1, eight crossbred dairy cows were inoculated with VACV-GP2 and in part 2, the cows from experiment 1 and 3 (part 1) were immunodepressed or reinfected. All animals in experiments 1, 2 and 3 were observed for 32 days and clinical examination was done every other day. Moreover, blood, faeces, oral swab and milk samples were collected daily and the technics employed were IPMA, seroneutralization and PCR. Several tissues were collected in necropsies and analysed through HE and IHC. Biochemical and hematological analysis and somatic cell count (SCC) in milk were done only in experiment 3. In all experiments it was observed that the incubation period was short and the evolution of the lesions was similar among the animals experimentally infected. Moreover, these lesions were also similar to the ones in cows naturally infected. Through clinical monitoring, it was observed that experimental infeccion of VACV in bovines causes local lymphadenopathy. Neither hyperthermy nor clinical alterations were detected. In the inoculated animals from experiments 1 and 2, histological alterations were observed in the teats, mammary glands and mammary lymph nodes in the three different phases of the disease, i.e., ulcerative, crust and healing. Through IHC, the VACV was detected in teats, as well as in mammary glands and mammary lymph nodes on the 3th, 9th and 17th d.p.i. During the experiment the mastitis was exacerbated by BV, which was shown by the increase of SCC in milk, and the dairy cows had intense decrease in milk production. lymphopenia and neutrophilia,which may be associated with viral infection and mastitis, respectively, were observed in the hematological analysis. In the experimental conditions which the cows were tested, it was possible to conclude that reinfection by VACV can occur in previously infected animals. There is a suspicion that the VACV can persist and multiply in immunodepressed bovines, once that there was an increase in antibodies titers in these animals. Lastly, some results suggest that the VACV can spread systematically in cattles, since the viral DNA was detected in the oral mucosa lesions.A presente dissertação foi dividida em três experimentos cujos objetivos foram reproduzir a vaccínia bovina (VB) em vacas secas e lactantes mestiças através da inoculação experimental com Vaccinia virus amostra GP2 (VACV-GP2). O objetivo do experimento 1 foi estudar a evolução clínico-patológica das lesões e a localização do vírus por imuno-histoquímica em períodos distintos da doença. Nesse experimento, seis vacas secas mestiças, divididas em três grupos, tiveram os tetos escarificados com auxílio de agulha hipodérmica em sítio delimitado, localizado na área central de cada teto. Dois grupos foram eutanasiados e necropsiados em fases diferentes de evolução da doença e em um grupo realizou-se apenas a biopsia dos tetos nos locais onde apareceram lesões. No experimento 2, três vacas mestiças foram inoculadas a fim de analisar o melhor método para escarificação de pele em tetos e a patogenicidade de dois inóculos diferentes contendo VACV-GP2. Uma vaca foi sacrificada no 17º d.p.i., período em que todos os tetos já haviam cicatrizado. Os objetivos do experimento 3 foram realizar os estudos clínico, hematológico e bioquímico associados à infecção e observar os efeitos da imunodepressão artificial e da reinoculação com VACV-GP2 em vacas lactantes previamente infectadas pelo vírus. Esse experimento foi dividido em duas fases, sendo que na fase 1, oito vacas mestiças em fases distintas de lactação foram inoculadas com o VACV e na fase 2, as vacas inoculadas nos experimentos 1 e 3 (parte 1) foram reinoculadas ou imunodeprimidas. Em todos os experimentos os animais foram acompanhados durante 32 dias e foi realizado o exame clínico em dias alternados. Além disso, foram coletadas amostras de sangue, fezes, suabe oral e leite e as técnicas realizadas foram IPMC, soroneutralização e PCR. Nas necropsias, foram coletados diversos tecidos para realização das técnicas de HE e IHQ. No experimento 3 foram acrescentadas as análises de bioquímica sérica e hemograma, além da contagem de células somáticas do leite. De maneira geral, observou-se que o período de incubação do VACV em bovinos é curto, e que o padrão de evolução das lesões ocorreu de maneira similar nos três experimentos, o qual também foi similar ao padrão de evolução de lesões observado em infecções naturais pelo VACV em bovinos. No acompanhamento clínico, foi observado que a infecção experimental do VACV em bovinos causa uma linfoadenopatia local e não foi detectada hipertermia nos bovinos experimentalmente inoculados, nem outras alterações clínicas nesses animais. Alterações histopatológicas significativas foram observadas nos tetos, glândulas mamárias e linfonodos retromamários dos animais inoculados dos experimentos 1 e 2, em três fases diferentes da doença: ulcerativa, crostosa e cicatrização. O VACV foi detectado, através da técnica de IHQ, nos tetos, glândula mamária e linfonodos retromamários dos animais da fase inicial (4º d.p.i) e da fase intermediária (9º d.p.i) da evolução da doença e na fase final, de cicatrização (17º d.p.i). Foi observado também que a mastite pode ser exacerbada pela VB e que houve queda de cerca de 30% na produção de leite nos animais infectados, assim como aumento significativo no número das células somáticas. Em relação ao perfil hematológico, foram observadas linfopenia e neutrofilia, que podem estar associados, respectivamente, com a infecção viral e a inflamação da glândula mamária. Mediante as condições experimentais às quais as vacas foram submetidas, foi possível concluir que a reinfecção pelo VACV pode ocorrer em animais previamente infectados. Suspeita-se também de que o VACV pode persistir e multiplicar em vacas imunodeprimidas, uma vez que houve aumento no título de anticorpos nesse grupo de animais. Por fim, alguns resultados do presente estudo sugerem que o VACV pode disseminar-se sistemicamente no organismo do bovino, uma vez que o DNA viral foi detectado nas lesões de mucosa oral.
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Law, Mansun. "Vaccinia virus spread : the roles of virus proteins, antibody and complement." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365358.
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Frischknecht, Friedrich. "Vaccinia virus mimics receptor tyrosine kinase signalling to achieve actin based motility." [S.l. : s.n.], 2000. http://www.diss.fu-berlin.de/2000/72/index.html.
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Xing, Xuekun. "DNA replication and telomere resolution in vaccinia virus." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq23557.pdf.
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Tseng, Michael. "Characterization of the vaccinia virus I3L gene product." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0003/MQ43227.pdf.
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Kenyon, Julia Claire. "The role of A41L in vaccinia virus immunogenicity." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424711.
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Haga, Ismar Rocha. "Characterisation of vaccinia virus genes A46R and A52R." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408267.
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Price, Nicola. "Characterisation of vaccinia virus genes B7R and B9R." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325962.
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Hiley, Crispin. "Arming vaccinia virus for pancreatic cancer oncolytic virotherapy." Thesis, Queen Mary, University of London, 2011. http://qmro.qmul.ac.uk/xmlui/handle/123456789/2344.
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Vaccinia virus is a 250-300nm enveloped DNA virus from the poxvirus family and is used as a vector for oncolytic viral gene therapy. No unique cell surface receptor has been identified for Vaccinia virus and the reasons for its tropism for cancer cells are unclear. Pancreatic adenocarcinoma (PDAC) is resistant to conventional chemotherapy and typically contains areas that are profoundly hypoxic. We have investigated the utility of Vaccinia virus as a vector for targeting hypoxic regions in pancreatic adenocarcinoma, as other viral vectors have been found to replicate poorly in hypoxia. We found that cytotoxicity was equivalent in normoxia and hypoxia in some PDAC cell lines but in others cytotoxicity was enhanced in hypoxia. This increase in cytotoxicity was only seen in cell lines where there was hypoxic induction of vascular endothelial growth factor (VEGF). Functional studies using over-expression and knockdown of VEGF in pancreatic cancer cell models showed that VEGF can augment viral transgene expression, cytotoxicity and replication in vitro and in vivo. We found that VEGF facilitates the internalisation of Vaccinia virus. These results show that VEGF is an additional factor involved in the tropism and pathogenesis of Vaccinia virus. We then constructed an oncolytic Vaccinia virus to target hypoxic cancer cells using the HIF-1α oxygen degradation domain, encephalomyocarditis virus internal ribosomal entry site and the VEGF 3‟ un-translated region to regulate luciferase expression in hypoxia. We have shown a dose-, time- and oxygen-dependent effect using this construct and propose this may be adapted to regulate therapeutic genes, or produce a conditionally replicating Vaccinia virus, in hypoxic conditions.
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Procter, Dean Joseph. "Genetic contribution to cytopathic effect in Vaccinia virus." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/12502.
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Vaccinia virus (VACV) infection induces cell migration, the formation of cytoplasmic extensions, actin polymerisation, cell rounding, detachment and lysis—changes collectively known as cytopathic effects (CPEs). Identification of genes that contribute to CPE phenotypes may highlight important virulence factors or novel signaling processes during infection. This investigation has focused on characterising four VACV genes (A55R, C2L, F3L and F5L) that have been identified as contributors to CPE. VACV protein F5 is a predicted membrane protein that is truncated in modified virus Ankara (MVA), a highly mutated strain with reduced CPE. We have characterised F5 to contribute to the rate of viral plaque expansion through a monolayer of infected cells, a contribution that may be derived from a localisation to cell junctions. The VACV BTB-BACK-Kelch proteins (BBKs) A55, C2 and F3 have been identified as contributors to CPE, with both A55 and C2 completely absent from the MVA genome. Human BBKs are well characterised as Cullin-3 substrate adaptors that facilitate the ubiquitination and subsequent degradation of various protein targets via the ubiquitin-proteasome system (UPS). Ubiquitination also has diverse non-degradative regulatory functions. Poxviruses are the only viruses known to encode BBKs and have been characterised to interact with Cullin-3. Our investigation has revealed functional redundancy between A55 and C2 from BBK-deficient CPE phenotypes. We have also been able to highlight a possible interaction of overexpressed VACV BBKs with ubiquitin-associated autophagy, a link between overexpression of C2 and filopodia formation and an association of endogenous expression levels of A55 and F3 with regulatory processes in the nucleus. Furthermore, we have been able to characterise the interactions of the VACV BBKs with each other, revealing that an A55:C2 heterodimer is most likely responsible for the CPEs generated by the VACV BBKs.
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McKenzie, Christopher David. "Activation of oncogenic signalling pathways by vaccinia virus." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15789.
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Vaccinia virus (VACV) is a dsDNA virus and a member of the Poxviridae family. VACV induces a wide range of morphological changes to cells during infection, collectively known as cytopathic effect (CPE). VACV-induced CPE includes loss of cell–cell contacts between neighbouring cells, induction of cell migration and extensive remodelling of the actin cytoskeleton in order to aid viral egress. These characteristic changes during VACV infection closely resemble the changes associated with epithelial-mesenchymal transition (EMT) — an important process in development, wound healing and cancer progression. EMT is controlled by multiple signalling pathways, with three of the most well-characterised pathways being the Wnt, TGFβ and Notch. We hypothesised that these same signalling pathways may also be involved in inducing the EMT-like changes observed during VACV infection. Using a luciferase reporter assay, we show that VACV activates TGFβ/Smad signalling but not Wnt/β-catenin signalling. Using both an inhibitor of the type-I TGFβ receptor and a cell line that lacks expression of the TGFβ receptor we found that VACV activates Smad signalling independent of receptor activity. We found that activation of Smad signalling is common to multiple strains of VACV but not the highly attenuated MVA strain or the related poxvirus, ectromelia virus. We identify and test several candidate VACV genes for their role in activation of Smad signalling and subsequently describe efforts to map candidate Smad-activating genes by partial rescue of the MVA genome. Finally, we aim to examine the contribution of Smad signalling activation to plaque phenotype in VACV. By adding TGFβ to exogenously activate Smad signalling in ECTV we found that activation of TGFβ signalling increases plaque clearance and size in ECTV plaques, as well as further increasing the size of VACV plaques, suggesting a role for activation of TGFβ/Smad signalling in the spread of infected cells within plaques.
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Pfanzelter, Julia. "The role of septins during vaccinia virus spread." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10042029/.
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Septins are highly conserved components of the cytoskeleton found in animals and fungi. They play a variety of roles in key cellular processes including cell division, cell migration and membrane trafficking. During host-pathogen interactions, septins inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. In addition, two recent genome-wide RNAi screens demonstrated that septins play an undefined role during vaccinia virus replication. Utilizing cell-based assays and microscopy I set out to determine the role of septins in vaccinia infected cells. I found that septins are recruited to vaccinia virus immediately following its fusion with the plasma membrane during viral egress. Live cell imaging reveals that septins are lost from beneath the virus once the virus stimulates Arp2/3 complex-dependent actin polymerization to enhance its cell-to-cell spread. Virus-induced actin polymerization involves the phosphorylation of the viral protein A36, leading to the recruitment of Cdc42, Nck, Grb2, WIP and N-WASP, which activate the Arp2/3 complex. Chemical or genetic inhibition of A36 phosphorylation dramatically increases the number of virus particles co-localizing with septins. Further experiments demonstrate that the recruitment of Nck and subsequently dynamin, but not Grb2, WIP:N-WASP or the Arp2/3-complex, promote the loss of septins from virions. RNAi-mediated depletion of septins increases virus release, accelerates cell-to-cell spread, and induces more robust actin tails. Collectively, my results demonstrate that septins limit the spread of vaccinia infection in cell monolayers and the recruitment of dynamin downstream of Nck enables the virus to overcome septin-mediated restriction. This is the first example of septins having an anti-viral effect and my work identifies a new role for septins in host defence.
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Wanas, Essam A. "Cloning and expression of the glycoproteins of pichinde virus by vaccinia virus." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6489.
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Pichinde virus (Pic), like the other arenaviruses, possesses two glycoproteins, GP1 and GP2, that are derived by proteolytic cleavage from a precursor molecule, GPC. Within the arenaviruses, GP1 is the most heterogeneous protein, and GP1 of Pic differs from that of the other arenaviruses in having twice as many potential N-linked glycosylation sites, most of which appear to be utilized. In order to examine the effects of this heavy glycosylation on Pic GP1 structure and inmunogenicity, GPC of Pic was cloned and expressed in vaccinia virus. The recombinant vaccinia (vvGPC) expresses authentic Pic GPC as demonstrated by immunoprecipitation with MAb and several polyclonal anti-Pic sera. GPC expressed in vaccinia is fully glycosylated as it comigrates with Pic GPC. At the same time, sequence analysis of cDNA shows both nucleotide and amino acid changes compared to published sequences for Pic GPC, indicating that variation in the same strain of this virus occurs as virus is passaged in separate laboratories. Experiments to assess the ability of Pic GPC expressed in vaccinia to elicit anti-Pic antibody show that rabbit anti-vvGPC detects authentic Pic GPC and GP1. Site-directed mutagenesis was employed to remove a potential N-linked glycosylation site (aa 181-183) in Pic GPC. Attempts were made to produce recombinant vaccinia, vvGPC-183, harbouring the mutant Pic GPC.
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Pelin, Adrian. "Bio-Engineering Vaccinia Viruses for Increased Oncolytic Potential." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39909.
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Vaccinia virus has a large and still incompletely understood genome although several strains of this virus are already in clinical development. For the most part, clinical candidates have been attenuated from their wild type vaccine strains through deletion of metabolic genes like the viral thymidine kinase gene. In the present work, we thoroughly examined the genetic elements of vaccinia which could be modulated to improve tailor the virus as a cancer therapeutic. Using a variety of cancer cell lines and primary tumor explants, we performed a fitness assay that directly compares multiple wild-type Vaccinia strains to identify the genetic elements that together create an optimal “oncolytic engine”. Using a transposon insertion strategy and deep sequencing of viral populations we systematically examined Vaccinia genes that do or do not play a role in the therapeutic activity of the virus. Our studies allowed us to identify a variety of genes in the vaccinia genome that when deleted, augment the oncolytic activity of a newly engineered Vaccinia virus. In the context of this thesis, I define enhanced oncolytic activity as superior therapeutic activity, increased immunogenicity and an improved safety profile, all aspects which we used to compare this novel virus to Vaccinia viruses currently in the clinic.
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Becker, Michelle Caitlin. "The Combination of Carboxylesterase-Expressing Oncolytic Vaccinia Virus and Irinotecan." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23653.
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This project combines oncolytic Vaccinia virus (VV) with irinotecan (CPT-11) for the treatment of cancer. VV can infect, replicate in and destroy cancer cells, yet leave healthy cells relatively unaffected. CPT-11 is a chemotherapeutic of which ~5% is converted to the more active chemotherapeutic SN-38 by endogenous carboxylesterase (CE) enzymes. SN-38 is a topoisomerase I inhibitor that induces DNA double strand breaks, leading to growth arrest and apoptosis. Consequently, VV has been engineered to express a more effective isoform of the CE enzyme. The virus’ tumour tropism should restrict enhanced conversion of CPT-11 to the tumour.
Neither CPT-11 nor SN-38 interfered with VV replication or spread. Engineered recombinants expressed CE enzyme which, when combined with CPT-11, produced DNA double strand breaks and cancer cell death. In vitro, the combination of CE-virus and CPT-11 killed more K-562 cancer cells than its non-CE counterpart and CPT-11.
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Roberts, Kim Louise. "The egress and entry of extracellular enveloped vaccinia virus." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444574.
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Cordeiro, Joao. "Modulation of Rho GTPase signalling during vaccinia virus infection." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/17273/.
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Infection by vaccinia virus leads to reorganization of the actin cytoskeleton, changes in cell adhesion, loss of contact inhibition and stimulation of cell motility. Previous work by the group has led to the identification of a viral gene, F11L, which is highly conserved among the orthopoxvirus genomes and is essential for this virus-induced cell mobility. F11L has been shown to interact with the RhoA but does not interact with the two other 'classical’ members of the family Rac1 and Cdc42. Understanding the molecular interaction of F11L and RhoA as well as investigating additional Rho binding partners was the basis of my thesis project with a view to obtain additional insights into the role of Rho GTPase manipulation during the vaccinia life cycle. Deletion of F11L from the virus genome results in reduced virus release and smaller plaque sizes. Characterization of the interaction between F11L and RhoA revealed that it occurs via a region of limited sequence homology to the RhoA effector ROCKI, in the C-terminal part of the molecule. Interestingly, generation of a recombinant mutant virus (F11L-VK) that is unable to bind RhoA leads to an intermediate phenotype between ΔF11L and WR in terms of viral plaque size. Further bioinformatic analysis revealed a region in the N-terminus of F11L with limited sequence homology to hDia2C, a RhoD effector. Biochemical analysis led to the identification of this site as the RhoD binding domain in F11L. Importantly, this study revealed that F11L interacts directly with different Rho GTPases via unique motifs to induce cell motility and facilitate enhanced virus cell-to-cell spread. Furthermore, work done in collaboration with the laboratory of Mariano Esteban in Madrid using a mouse model of infection shows that in contrast to wild-type vaccinia, infection with ΔF11L does not result in animal death. Additionally, the F11L-VK virus shows limited spread in vivo but still induces animal death albeit at a later stage when compared to wild-type vaccinia. These results suggest that F11L has additional functions besides interacting with RhoA, consistent with my observations that it can interact with other Rho GTPases. These studies can provide valuable information about the importance of Rho GTPase manipulation for the life cycle of vaccinia virus but also indicate the potential for the genetic manipulation of F11L in existing poxvirus vectors that may improve their therapeutic potential, safety, immunogenicity and/or oncolytic activity.
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Weisswange, Ina. "Analysis of Vaccinia virus actin tail nucleating complex dynamics." Thesis, University College London (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499526.
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Price, Philip John Ritchie. "Leukocyte trafficking during infection with modified vaccinia virus Ankara." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-173737.
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Whilding, Lynsey May. "Activity of oncolytic vaccinia virus vectors in ovarian cancer." Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8553.
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Oncolytic vaccinia virus has great potential in the treatment of cancer and two engineered strains have entered clinical trials. As the advent for oncolytic vaccinia virus as an approved therapy beckons, it is critical to consider some of the barriers that may hinder this progress. These include suboptimal delivery of the virus to tumour sites, incomplete destruction of the tumour mass, and a lack of full understanding of the way in which oncolytic vaccinia kills its target cells. This thesis attempts to address these issues, with a particular focus on ovarian cancer. As ovarian cancer is generally restricted to the peritoneal cavity, intraperitoneal delivery may be preferable over intravenous delivery. Here, it is shown that Lister-dTK, an engineered vaccinia strain, is able to selectively replicate in ovarian tumours, including metastases to the liver following intraperitoneal delivery. To determine whether Lister-dTK could potentially be used in combination with current therapies for ovarian cancer, the effect of cisplatin and Lister-dTK together was assessed in vitro but showed no improvement in overall cell death. In an attempt to further improve the anti-tumour efficacy of Lister-dTK, the extracellular matrix protein (ECM) decorin was expressed from the virus. Decorin interacts with various signalling pathways and is proposed to enhance virus spread. However, abrogation of EGFR and TGFβ signalling could not be demonstrated in vitro, nor could improved virus spread. In an intraperitoneal model of ovarian cancer, Lister-mDCN did not demonstrate enhanced efficacy over a control virus. To determine the mechanisms of ovarian cancer cell death induced by Lister-dTK, the roles of apoptosis, autophagy and necrosis were investigated. Whilst some features of both apoptosis and autophagy were observed, inhibition of these pathways did not attenuate Lister-dTK. It is proposed that necrosis is the primary cause of cell death but that this process may occur in a regulated manner.
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Gao, William Ning Da. "Viral and cellular proteins involved in vaccinia virus egress." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/280280.
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Vaccinia virus (VACV) is a large double-stranded DNA virus with a cytoplasmic site of replication. It has a complex life cycle that produces two distinct infectious virion forms, Intracellular Mature Virions (IMVs) and Extracellular Enveloped Virions (EEVs). The host cell microtubule trafficking machinery is hijacked by the virus at three distinct positions of the viral life cycle. After virus entry, the virus cores are transported to pre-nuclear sites where they form viral factories that ultimately produce fully functional and infectious IMVs. A small proportion of IMVs are further transported to sites of wrapping, where they are enveloped by a host-derived double membrane to form Intracellular Enveloped Virions (IEVs). The IEVs are then transported to the cell periphery to facilitate efficient viral spread. The viral proteins A36, F12 and E2 together with the kinesin-1 microtubule motor protein are thought to be involved in IEV egress from the site of wrapping to the cell periphery, although the exact mechanism of movement is unclear. Until recently, A36 was the only known protein to interact with the kinesin-1 motor through kinesin light chain (KLC), but F12 has also been shown to interact with KLC through E2. The precise mechanism of how the IEV interacts with and activates the kinesin-1 motor protein is unclear, and this study explores the interactions of IEV proteins with KLCs in detail, mapping interactions between KLC and A36 or F12/E2. A36, F12 and E2 also show no sequence or predicted structural homology to any other known proteins, and structural studies were performed in an attempt solve their 3D structure. The CRISPR-Cas9 targeted genome editing tool was also utilised to knockout different KLC isoforms in multiple cell lines to assess their contribution to IEV egress as well as cellular trafficking. These studies will provide insight into the mechanisms behind the spatial and temporal control of kinesin motor activity in the cell.
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Dave, Rajiv Vipool. "Oncolytic vaccinia virus for the treatment of liver cancer." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/6887/.
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Aims: Current treatment of colorectal cancer (CRC) liver metastases has a success rate of 50% 5-year survival, and recurrence rates of 50%. There is therefore still a need for a novel treatment modality. We aimed to examine the ability of JX-594 to preferentially replicate in and kill CRLM in vitro and ex vivo, and induce immunemediated tumour cytotoxicity by activation of natural killer (NK) cells. Methods: The Wyeth strain of vaccinia virus has been genetically manipulated to encode for granulocyte macrophage colony stimulating factor (JX-594-GM-CSFfLuc) and green fluorescent protein (JX-594-GM-CSF-GFP) in the disrupted thymidine kinase locus. Viability assays and Enzyme Linked Immunoabsorbent Assay (ELISA) was used to confirm tumour cell killing and production of inflammatory cytokines when CRC cell lines were infected with JX-594. Viral replication in vitro was investigated by plaque assay and using an ex vivo ‘tissue core’ method. Induction of the innate immune response was measured by upregulation of activatory markers on virus-treated-NK cells and monocytes by flow cytometry and anti-tumour cytotoxicity by chromium release. Results: JX-594 can directly lyse CRC cell lines, with greater lysis and replication (up to 250-fold) in cells with upregulated surface EGFR. JX-594 treatment resulted in substantial expression of GM-CSF and induction of inflammatory cytokines within the tumour microenvironment, and inhibition of anti-inflammatory and proangiogenic cytokines. Ex vivo infection of CRLM with JX-594-GFP-GM-CSF resulted in tumour-specific GFP and GM-CSF expression. Treatment of NK cells with JX-594-GM-CSF led to activation, degranulation and increased cytotoxicity against CRC cell targets. This was dependent on the presence of CD14+ve monocytes, which acquired an antigen-presenting phenotype (CD86+veCD11c+veClassIIDR+ve). Conclusions: JX-594 holds promise as a novel treatment modality for disseminated CRC. Direct tumour-specific lysis and transgene expression and the induction of tumour-specific innate immunity means that it may provide a twopronged attack against tumour cells whilst sparing normal tissue.
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Laporte, Aimée N. "Enhancing the Oncolytic Efficacy of Vaccinia Virus by Mutagenic Augmentation of EEV Production." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23348.
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Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.
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Dunstan, Kerrie Women's & Children's Health Faculty of Medicine UNSW. "Understanding the early interactions between vaccinia virus and dendritic cells - towards an enhanced vaccine vector." Awarded by:University of New South Wales. Women's and Children's Health, 2007. http://handle.unsw.edu.au/1959.4/32456.
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In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.
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Scheubeck, Gabriel Maria [Verfasser]. "Starvation-Induced Differential Virotherapy Using an Oncolytic Measles Vaccine and Vaccinia Virus / Gabriel Maria Scheubeck." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1226756654/34.
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Sullivan, Veronica. "Analysis of the herpes simplex virus type 1 glycoproteins using recombinant vaccinia virus vectors." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292957.
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Veyer, David. "Comparaison des propriétés antiapoptotiques de quatre protéines du virus de la vaccine en isolement et au cours de l’infection virale." Thesis, Rennes 1, 2014. http://www.theses.fr/2014REN1B015/document.
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L’apoptose, mort cellulaire observée suite à l’activation des caspases effectrices, est un moyen de défense contre les pathogènes, en particulier les virus. Le virus de la vaccine (VACV) est un virus contenant un grand génome à ADN codant pour environ 200 protéines, dont plusieurs inhibent l’apoptose. Cette apparente redondance fonctionnelle complique l’étude des protéines antiapoptotiques du virus dans un contexte d’infection virale. Dans ce travail, nous comparerons les propriétés antiapoptotiques des protéines B13, F1, GAAP et N1 de VACV. Cette comparaison sera établie dans un premier temps en dehors de toute infection virale. En utilisant des vecteurs lentiviraux, nous avons obtenu des lignées cellulaires stables (U2-OS) exprimant ces protéines en isolation. Nous avons alors pu tester les capacités antiapoptotiques de ces protéines en réponse à des stimuli provoquant l’apoptose extrinsèque et intrinsèque. Les résultats ont montré que B13 était la plus puissante molécule inhibitrice de l’apoptose intrinsèque et qu’elle était la seule à inhiber l’apoptose extrinsèque. Ensuite nous avons tiré avantage d’un virus de la vaccine déficient (vv811) qui ne possède aucune de ces protéines antiapoptotiques, capable à lui seul d’induire l’apoptose, en l’absence de toute autre stimulus. En infectant nos lignées cellulaires exprimant les molécules in trans avec vv811, nous avons pu montrer que B13 inhibait cette apoptose induite par le virus beaucoup plus efficacement que F1. GAAP et N1 dans ce contexte n’ont pas démontré de propriétés antiapoptotiques. Enfin, nous avons construit par mutagénèse des virus vv811 recombinants exprimant les molécules étudiées in cis. Suite à l’infection par ces virus de cellules U2-OS et Hela, B13, de nouveau, et F1 ont montré des capacités d’inhibition importantes de l’apoptose. L’action de GAAP s’est révélée dépendante du type cellulaire et N1 n’a pas pu inhiber l’apoptose induite par ce virus déficient dans aucune des cellules testées. En utilisant ces différentes approches, nous avons pu nous affranchir des problèmes de redondance et comparer 4 molécules antiapoptotiques du virus de la vaccine, y compris dans un contexte d’infection virale. Les résultats ont confirmé que toutes les protéines étudiées possédaient des propriétés antiapoptiques et ont clairement montré que B13 était la plus puissanteApoptosis, which occurs following activation of effector caspases, can restrict the replication of intracellular pathogens, especially viruses. Vaccinia virus (VACV) is a large dsDNA virus encoding approximately 200 proteins, several of which inhibit apoptosis. This redundancy of viral anti-apoptotic proteins complicates the study of these proteins in the context of viral infection. Here a comparative study of the anti-apoptotic proteins B13, F1, GAAP and N1 with and without virus infection is presented. Firstly, using lentiviral constructs, we generated transduced cell lines expressing the anti-apoptotic proteins in isolation and we analysed their ability to protect against extrinsic and intrinsic apoptosis induced by different drugs. In that context B13 was the most potent inhibitor of intrinsic apoptosis and the only protein to inhibit both extrinsic and intrinsic apoptosis. We then used a deficient VACV strain, vv811, that lacks the genes coding for the four anti-apoptotic proteins. Infection with vv811 can induce apoptosis without the need for any other stimulus. After vv811 infection of cell lines expressing the anti-apoptotic proteins in trans, B13 and to a lesser extent F1, inhibited apoptosis. Finally, we introduced each gene separately into vv811 by genetic recombination. Using these recombinant viruses to induce apoptosis, B13 and F1 were very potent inhibitors. The protection conferred by GAAP was cell type dependant and N1 failed to protect any of the tested cells from the virus induced apoptosis. Using these different approaches, we have been able to overcome the redundancy issue to compare 4 anti-apoptotic proteins from VACV, including in the context of viral infection. The results illustrate that vv811 is a useful tool to determine the role of VACV anti-apoptotic proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 is most potent
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Cresawn, Steven Gaines. "Genetic inquiry into vaccinia virus intermediate and late gene regulation." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0010102.
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Thesis (Ph.D.)--University of Florida, 2005.Typescript. Title from title page of source document. Document formatted into pages; contains 150 pages. Includes Vita. Includes bibliographical references.
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MacTavish, Heather L. "Enhancement of Vaccinia Virus Based Oncolysis with Histone Deacetylase Inhibitors." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28659.
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Histone deacetylase inhibitors (HDI) dampen cellular immune response by decreasing interferon production and have been shown to increase the replication of Vesicular Stomatitis Virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective VV in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet TSA treatment did not increase the replication of VV in normal tissues. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells.
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Kent, Richard Keith. "Isolation and analysis of the vaccinia virus P4B gene promotor." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303279.
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Ahmad, Khawaja Muneer. "Mutagenesis and localisation studies on the vaccinia virus B7R protein." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405439.
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Van, Eijl Henriette Joselijn Leonie. "The distribution and topology of the vaccinia virus A36R protein." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325640.
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Scutts, Simon Robert. "Investigations into the vaccinia virus immunomodulatory proteins C4 and C16." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/275923.
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Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.
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Evans, Elizabeth Van Amburg. "Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication /." Access full-text from WCMC, 1989. http://proquest.umi.com/pqdweb?did=744576211&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Baloglu, Simge. "Applicability of vaccinia virus as cloning and expression vector for bacterial genes: mice immune responses to vaccinia virus expressing Brucella abortus and Listeria monocytogenes antigens." Diss., Virginia Tech, 2001. http://hdl.handle.net/10919/28485.
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Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1).
As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge.
Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge.
Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant.Ph. D.
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Griffiths, Caroline Mary. "The cloning and expression of Herpes simplex virus type 1 glycoprotein C in vaccinia virus." Thesis, University of Leicester, 1991. http://hdl.handle.net/2381/35382.
Full textAbstract:
The Herpesviridae family contains numerous virus types, several of which can infect man. The virus which is most widely spread in terms of infection is herpes simplex virus (HSV), of which there are two serotypes (HSV-1 and HSV-2). Following a primary infection, the virus can establish a latent infection within neurons of sensory ganglia, where it remains throughout the lifetime of the host. HSV is able to reactivate from the latent state, and may produce clinical infection at the site of the initial virus invasion (recrudescent lesions). Although most HSV-1 infections are mild or subclinical, infection can lead to life-threatening illness. The search for an effective HSV vaccine has met with limited success to date. In recent years, vaccine research has turned toward the use of viral vectors, for the expression of individual virus proteins with a view to stimulating host immunity. A great deal of attention has been focussed on the vaccinia virus, which is able to accept large amounts of foreign DNA into it's genome with no loss of viability. Several HSV proteins have been expressed from recombinant vaccinia viruses, and this project outlines the cloning of DNA sequences encoding the HSV-1 glycoprotein C, and expression of that protein from a recombinant vaccinia virus. The HSV DNA sequences encoding the glycoprotein were cloned from an existing library, into plasmid vector pSC11. This plasmid allows insertion of gC sequences into the vaccinia virus genome by way of homologous recombination. The plasmid also provides a vaccinia virus promoter for the control of gC transcription during infection of cells with the resulting recombinant viruses. Recombinant vaccinia viruses produced on cloning of a 1.75kb NheI/SphI fragment containing the HSV-1 gC gene did not express the glycoprotein during infections. Vaccinia virus DNA polymerase is known to be sensitive to secondary structures within DNA, and the 5' non-coding region of the HSV gC gene is rich in GC basepairs, which could readily form such structures. Site-directed mutagenesis removed the 5' non-coding region (34 nucleotides) in an attempt to remove any such block to gC transcription. Vaccinia viruses containing the mutated sequences were able to express gC within infected cells, as determined by immunofluorescence. Mice inoculated with a gC-expressing recombinant vaccinia virus were able to induce HSV-neutralising antibodies and displayed 50% protection against a lethal HSV-1 challenge. The implications of gC-expressing vaccinia recombinant viruses, with respect to the search for an HSV vaccine and to the understanding of the role of gC during HSV infections is discussed.
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Nie, Siwei. "Role of TNF in Heterologous Immunity between Lymphocytic Choriomeningitis Virus and Vaccinia Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/394.
Full textAbstract:
Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system.
TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV.
We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications.
One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease.
Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host.
The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.
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Alharbi, Naif K. "New approaches for improving the immunogenicity of modified vaccinia virus Ankara as a recombinant vaccine vector." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:bbde86fd-ea8f-4e66-b260-f923d7e01e4b.
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