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Strang, Blair Lewis. "Development and characterisation of pseudotype HIV vectors." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406402.
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Piccini, Giulia. "Evaluation of Influenza Lentiviral Pseudotypes as an alternative source of antigen in the Neuraminidase Inhibition Enzyme-Linked Lectin Assay." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1096232.
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The continuously changing epidemiology of influenza viruses needs to be yearly addressed with a fast and massive production of vaccines that prevent epidemic outbreaks and occasional pandemics worldwide. Currently licensed flu vaccines are designed to elicit antibodies against the viral hemagglutinin (HA). Formulations containing standardized amounts of neuraminidase (NA) are however gaining growing interest in the influenza vaccine field, as NA‐inhibiting (NI) antibodies have been associated with resistance to disease as well as reduced severity and duration of illness.
The Neuraminidase Inhibition Enzyme-Linked Lectin Assay (NI-ELLA), which quantifies NI antibody titers based on the ability of specific antibodies to inhibit NA activity, is regarded as a reliable and high-throughput method to measure NI antibodies in human sera. To avoid the non-specific inhibition of HA-binding antibodies that may be present in serum samples, viruses containing an antigenically mismatched HA (reassortants) or Triton-X100(Tx)-treated wild-type (WT) viruses that retain NA functionality have been employed as NA source in the ELLA assay. An innovative approach that exploits NA-bearing pseudotypes (or pseudoviruses, PV) as surrogate viruses expressing the NA of interest has been recently investigated to provide a safer product to be used for the ELLA test.
The first aim of this PhD work was to evaluate three different sources of NA (reassortant viruses, Tx-inactivated WT viruses, and lentiviral PV) to compare their ability in the measurement of NI-ELLA titers. In order to do that, two independent panels of sera were analyzed by ELLA assay to investigate the antibody response against influenza NA subtype N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NI titers were measured either as the 50% endpoint or 50% inhibitory concentration (IC50) and were compared for every source of antigen. The linear regression, Spearman rank, Bland-Altman and Intraclass Correlation (ICC) analyses were employed to assess the comparability of the data obtained.
Analysis of results showed that the ELLA performed well with all three sources of NA. The correlation coefficients (r2) for all comparisons were high (r2 > 0.92) when titers were reported either as endpoint or as IC50. Comparisons that included PV as antigen source demonstrated the best concordance, with 90% of N1 titers and 88% of N2 titers measured by the pseudotypes-based ELLA (PV-ELLA) showing less than 2-fold difference compared to those obtained in the assays performed with the other sources of NA. ICC results showed a high concordance between the different methods when either examining N1 or N2 titers (ICC coefficients of 0.861 and 0.817, respectively). Bland-Altman analyses revealed an overall agreement, and showed that titers measured against N2 antigens (reassortant H6N2 and H11N2 PV) were in better agreement than N1 titers measured using reassortant H6N1 and H11N1 PV antigens. For both anti-N1 and anti-N2 titers, the assays performed with the available sources of antigen show similar ability to detect NI titers ≥40, which have recently been associated with protection against influenza. The different ELLA platforms show comparable Geometric Mean Titer (GMT) values as well as similar titers range, suggesting the suitability of performing ELLA with each of the NA sources evaluated.
The second aim of this project was to validate the PV-ELLA executed by using H11N1ACal/09 and H11N2HK/14 PV, following the recommendations provided by international guidelines. Both the ELLA performed with H11N2HK/14 PV and H11N1ACal/09 PV fulfill the pre-established criteria for definition of assay specificity, as the titer raised by the high positive serum sample against the homologous PV was always ≥ 4-fold higher than that of the heterologous and negative samples, which always displayed undetectable NI responses.
The N1 and N2 PV-ELLA showed a strong linear trend between dilutions of the reference sample vs the respective GMT in the range of serum concentrations evaluated, with r2 coefficients always higher than 0.96 and 95% confidence intervals of the slope within the acceptability range, suggesting excellent linearity of the assay. The Geometric Mean Titers (GMTs) yielded in these linearity experiments show a maximum deviation of ± 1 titer step dilution (i.e. ≤ 2-fold difference in titers) when (i) collected within the same day or in different days, and when (ii) compared to those obtained in a pre-validation testing, suggesting precision and accuracy of the PV-ELLA. None of the variations applied to three critical steps of the assay produced a significant impact on NI titers, indicating that the ELLA performed with H11N1ACal/09 and H11N2HK/14 PV is adequately robust.
In summary, the PV-ELLA performed with either N1- and N2-bearing PV has met all the acceptability criteria pre-defined for demonstration of specificity, linearity, precision, accuracy and robustness of the analytical method, confirming that the assay is suitable to measure NA antibody response in the experimental conditions evaluated.
These results support the use of lentiviral pseudotypes as an attractive alternative source of NA in the ELLA assay and provide the means to standardize and validate the methodology in laboratories that do not have access to suitable reassortant viruses.
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Mahoney, Catherine H. "Studies using pseudotyped retroviral vectors." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312721.
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Grzybowski, Brad. "A pseudotyped viral vector : hPIV3-HIV-1." Thesis, Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/20932.
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Thomas, Joan Helen. "Studies in gene transfer using pseudotyped lentiviral vector systems." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621818.
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Gaiano, Nicholas R. (Nicholas Roger). "Insertional mutagenesis in zebrafish using a pseudotyped retroviral vector." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/43311.
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Peng, Yue. "A novel vaccine strategy : replication-defective pseudotyped SIVs expressing IFN-[gamma] /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Thesis (Ph. D.)--University of California, Davis, 2005.Degree granted in Comparative Pathology. On title page "[gamma]" appears as lower-case Greek letter. Also available via the World Wide Web. (Restricted to UC campuses)
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Kinsley, Rebecca. "Development of lentiviral pseudotypes for surveillance studies on animal influenza viruses." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/66297/.
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Pseudotyped viruses (PVs) provide a safe, flexible platform for fundamental virological studies and antibody screening assays. Generation of influenza PVs involves co-transfection of producer cells with plasmids encoding the necessary viral components. The pseudotype virus neutralisation assay (PVNA) is a sensitive technique to measure protective antibody responses which cause neutralisation of virus particles. Many traditional methodologies, e.g. Haemagglutination Inhibition (HI) and Single Radial Haemolysis (SRH), detect only surface glycoprotein binding antibodies whereas the PVNA quantifies infectivity-neutralising responses. Haemagglutinin (HA) and neuraminidase (NA) are the two major surface glycoproteins embedded within the membrane of an influenza virion. HA is responsible for virion attachment and entry into a host cell and NA is essential for viral egress and thus spread of infection. Two enzymes crucial for the infectivity of influenza viruses are; HA-cleaving cellular proteases and the NA itself. Optimisation of both enzymes in PV production is necessary to increase the titre of PVs. Producing high titre PVs is important as this permits minimal quantities to be used in PVNAs, and repeat experiments can be carried out using the same batch of virus, minimising intra-study variability. Optimising PVs and employing them in a novel situation, such as an equine influenza vaccine efficacy trial, has been carried out with promising results. We have demonstrated that data obtained from the PVNA correlates well with the traditional SRH assay and consequently there is the potential for more widespread adoption in research and commercial settings. Furthermore, PVs have been manipulated to assess how single amino acid changes with equine influenza virus can affect the neutralisation efficacy of sera generated by vaccination. Novel PVs, such as those derived from canine and phocine (seal) influenza strains have also been produced and provide a new platform for sero-surveillance of these viruses particularly in the case of wild or feral animals, due to issues with obtaining samples from wild animals during acute infection. Overall, PVs have been demonstrated as useful and readily-manipulated tools for studying antibody responses against equine, canine and phocine influenza viruses.
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Carnell, George William. "Development of hybrid haemagglutinin pseudotyped lentiviruses to assess heterosubtypic immunity to influenza." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/66363/.
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The influenza virus still causes hundreds of thousands of deaths globally, on top of morbidity and associated economic burden. We are currently at the height of efforts surrounding the development and employment of 'universal' vaccines against this virus, with clinical trials commencing on the most promising candidates. Despite this, the influenza virus poses more of a threat to human life than it ever has previously, with multiple subtypes of pandemic potential circulating around the globe. The key to current efforts lies in the priming of the immune system towards generating long lasting defences against conserved epitopes and conferring heterosubtypic immunity against the surface glycoprotein haemagglutinin. While vaccine strategies have expanded rapidly over recent years with the advent of 'headless' constructs as well as those derived from consensus, mosaic or chimeric sequences, the serological techniques to test how effective these vaccines are, have advanced less rapidly. Classical serological assays have been shown to be ineffective at detecting the antibodies which modern 'universal' vaccines strife to elicit, replaced by ELISA based approaches combined with mouse models measuring in vivo protection. In this thesis, an alternative method for the detection of heterosubtypic antibodies is used in depth across multiple platforms. Influenza pseudotypes have been employed using chimeric haemagglutinin constructs in a comprehensive project aimed at dissecting head and stalk directed antibodies present in human serum. Characterised broadly neutralising monoclonal antibodies have been tested on panels of influenza pseudotypes including divergent bat influenza viruses which hitherto have not been encountered in humans. A further aspect of influenza immunity has been covered in the detection of anti neuraminidase antibodies which have an important role to play in influenza heterosubtypic immunity. Finally, influenza pseudotypes bearing the glycoproteins from the less studied influenza B virus have been assayed in a large scale project aimed at correlating pseudotype assays with classical approaches.
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Amsterdam, Adam (Adam Henry) 1967. "Use of a pseudotyped retoviral vector to accomplish insertional mutagenesis in zebrafish." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/50024.
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Ferrara, Francesca. "Using pseudotypes to study heterosubtypic antibody responses elicted by seasonal influenza vaccination." Thesis, University of Kent, 2015. https://kar.kent.ac.uk/50903/.
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Influenza viruses represent an important public health burden since they cause annual epidemics associated with severe illness and mortality in high-risk populations. Additionally, zoonotic influenza virus infections have potential to produce intermittent pandemics, which have led to millions of deaths globally. However, strategies to prevent influenza severity and spread can be implemented. It is known that antibodies against the haemagglutinin play a key role in protection from influenza virus infection, thus both seasonal and pandemic influenza vaccines aim to elicit such antibodies. Generally, they are directed against haemagglutinin globular head epitopes and are able to neutralize closely related influenza strains, but recently antibodies able to neutralize multiple influenza strains and subtypes have also been described. The discovery of these antibodies, primarily directed against the haemagglutinin stalk, has generated interest in understanding how they are generated and how widespread they are in the human population. Furthermore, eliciting such antibodies has become the aim of many ‘universal’ vaccine approaches. However, the study of these cross-reactive antibodies using classical serological assays is problematic since the current assays have been shown to be relatively insensitive in detecting them. The main objective of this thesis was to study the presence and breadth of cross-reactive neutralizing responses in human populations. To overcome the limitations of current serological tests in detecting these responses, lentiviral pseudotype particles bearing the haemagglutinins of different influenza A subtypes and influenza B strains were used as surrogate antigens in neutralization assays. After the generation of these novel tools and the establishment of appropriate controls, pseudotype particle neutralization assays were employed to investigate cross-reactive antibody responses in pre- and post-vaccination sera. Next, the use of chimeric haemagglutinins, in which the globular head was substituted with the head of a different subtype, was incorporated into the pseudotype system. This allowed the differentiation between haemagglutinin head-directed and stalk-directed antibody responses. The ability to efficiently detect broadly neutralizing antibody responses, including those directed against the haemagglutinin stalk, shows that pseudotype particles are tools that should be further characterised and implemented to be used in sero-epidemiological studies and for ‘universal’ vaccine immunogenicity studies.
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Mather, Stuart Thomas. "Development of pseudotyped virus assays for the serological study of Japanese encephalitis flavivirus." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/62936/.
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Japanese encephalitis virus (JEV) is one of the primary global causes of viral encephalitis, with approximately 68,000 clinical cases and 20,000 deaths attributed to the virus annually. Between 30% and 50% of survivors suffer from debilitating neurological sequelae. Despite being a vaccine-preventable disease, no antiviral treatments are licensed and commercially available to counteract JEV infection. In order to quantify the neutralising antibody response raised against antigenic epitopes on flavivirus prME glycoproteins, conventional serological assays such as the plaque reduction neutralisation test (PRNT) can be employed. However, these assays often necessitate the handling of pathogenic wild-type virus in expensive high-biosafety laboratories, restricting the scope of their application, particularly in resource-deprived areas. Chimeric, replication-deficient pseudotype viruses can offer a solution to this problem, as they mimic wild-type virus entry mechanisms, enabling their use in pseudotype virus neutralisation assays (PVNAs). PVNAs bypass high biosafety requirements and permit vaccine evaluation and serosurveillance studies with no risk of inadvertent infection. This project focuses on the production of functional pseudotype viruses displaying the prM and E surface glycoproteins of the JEV flavivirus, for utilisation in serological neutralisation assays. Subcloning of the prME gene into an appropriate eukaryotic expression vector and insertion mutagenesis to produce prME with 15- and 24-residue upstream signal peptides are shown, before production of JEVpp with either HIV or MLV cores is attempted, via the conventional multi-plasmid co-transfection approach or the utilisation of constitutive gag-pol expressing cell lines. The impact of additional plasmid-derived furin protease expression and low glucose culture medium, as well as the construction of JEV/VSV chimeric prME glycoproteins and the introduction of Kozak consensus sequences upstream of the prME gene, to enhance the efficiency of JEVpp generation is also explored. Finally, the infectivity of lentiviral pseudotype viruses following lyophilisation, storage and reconstitution is confirmed, thus enabling their affordable global distribution.
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Pfaff, Kerstin [Verfasser]. "Hepatitis-C-Virus-Pseudotypen und deren Einsetzbarkeit in der virologischen Diagnostik / Kerstin Pfaff." Berlin : Freie Universität Berlin, 2012. http://d-nb.info/1029850569/34.
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Enkirch, Theresa [Verfasser], and Ralf [Akademischer Betreuer] Bartenschlager. "Targeted lentiviral vectors pseudotyped with the Tupaia paramyxovirus glycoproteins / Theresa Enkirch ; Betreuer: Ralf Bartenschlager." Heidelberg : Universitätsbibliothek Heidelberg, 2011. http://d-nb.info/1179783220/34.
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Bentley, Emma. "The study of highly pathogenic emerging zoonotic virus envelope proteins through pseudotyped virus generation." Thesis, University of Westminster, 2017. https://westminsterresearch.westminster.ac.uk/item/q4yzx/the-study-of-highly-pathogenic-emerging-zoonotic-virus-envelope-proteins-through-pseudotyped-virus-generation.
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Emerging zoonotic viruses pose an increasing threat, causing outbreaks with high rates of morbidity and mortality and frequently significant economic implications. Often, there is a lack or shortfall of effective prophylaxis and diagnostic capabilities. Research towards their development, together with improved surveillance activities are high priority activities to prepare and respond to outbreak threats. Yet handling these viruses commonly requires high containment levels. This can be circumvented by the use of replication defective pseudotyped viruses (PVs), incorporating the viral envelope protein of interest which constitutes the primary surface antigen. This permits the serological detection of neutralising antibodies without the need to handle live virus, as well as other viral entry studies. Hence, PVs are increasingly proving to be a valuable tool for emerging virus research. The aim of this study was to exploit novelties in the unique flexibility of the PV platform to allow the serological assessment of emerging viruses and evaluate technical aspects towards standardisation. Current prophylaxis provides robust protection against rabies virus, yet only confers limited protection against other lyssavirus species, which have a near 100% fatality rate. It is thought protection is afforded against isolates of phylogroup I rabies virus, yet there is limited biological data for the Arctic-like rabies virus (AL RABV) lineage which is endemic across the Middle East and Asia. Although other lyssaviruses pseudotype efficiently, titres of AL RABV PV were low. Within this study, high titre PV was produced by constructing chimeric envelope proteins, splicing the AL RABV ecto-transmembrane domain with the cytoplasmic domain of vesicular stomatitis virus. Comparisons showed this did not alter the serological profile of the AL RABV and they were effectively neutralised by vaccines and antivirals. It could therefore be concluded that they do not pose a significant public health risk. However it is recognised broadly neutralising prophylaxis needs to be developed to protect against more divergent lyssaviruses. In a further study, again utilising the flexibility to manipulate the envelope protein, PV was produced switching the five known antigenic sites of the envelope protein between a phylogroup I (rabies virus) and III (West Caucasian bat virus) isolate. Screening polyclonal sera via a neutralisation assay, the immunologically dominant sites for phylogroup I and III were identified as III and I respectively. This can act to inform future development of more broadly neutralising vaccines. The 2013-16 outbreak of Ebola virus focused global efforts towards the urgent need for effective vaccines and antivirals. To permit low containment level serology studies to assist their development, a panel of filovirus PVs were rapidly produced. Work was carried out to optimise their method of production; determining lentiviral core PV produced by transfecting HEK 293T/17 cells was most efficient. Efforts to repeat the use of chimeric envelope proteins to increase titre proved unsuccessful. The evaluation of target cell lines permissive to infection and appropriate for neutralisation assays identified that the CHO-K1 cell line produced the clearest data. The PV neutralisation assay was subsequently applied to a range of projects to assess candidate prophylaxis and demonstrated the value of the platform to respond to emerging virus outbreaks. Given the increasing prominence in the use of PV, work was undertaken to expand their utility and methods for standardisation. An assessment of new reporter genes found a red fluorescent protein, with a nuclear localisation signal, improved the clarity of data collection and output in additional spectrum to the current repertoire. To be able to correlate the disparate readout units of fluorescent and luminescent reporters, recorded as infectious units (IFU) and relative light units (RLU) respectively, a new construct was produced to integrate and equally express two reporters from cells transduced with PV. It was determined that approximately 1260 RLU equates to 1 IFU, although future work to determine how this fluctuates between cell lines is required. Finally, alternative methods to quantify PV were evaluated, measuring the number of particles, genome copies and reverse transcriptase (RT) activity, in addition to the currently used biological titre. It was found that measures of genome copies and RT activity, in combination with biological titre provides information on the quality of PV preparations and could be used to standardise assay input.
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Mao, Jian. "Vesicular stomatitis virus G pseudotyped retrovector mediated gene delivery of connexin43 to brain tumor cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0001/MQ42173.pdf.
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Haynes, Soraya. "Construction of a novel bat coronavirus glycoprotein pseudotyped lentiviral vector and analysis of cell tropism." Thesis, Haynes, Soraya (2019) Construction of a novel bat coronavirus glycoprotein pseudotyped lentiviral vector and analysis of cell tropism. Honours thesis, Murdoch University, 2019. https://researchrepository.murdoch.edu.au/id/eprint/54215/.
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Since the SARS epidemic in 2003, and the subsequent identification of bats as the reservoir host, there has been a surge of interest in research into bat-borne viruses, with over 30 new bat coronaviruses alone being discovered. Investigating the cell tropism of these newly discovered coronaviruses deepens the understanding of their pathology and possible host ranges, as well as allowing assessments to be made on their zoonotic potential. Aside from biosafety concerns however, this is also reliant on isolation of the virus from its host, which can be difficult using conventional methods.
This project aimed to develop a second generation lentivirus pseudotyping system which would allow spike proteins, the main determinants of coronavirus cell entry, of a non-isolated coronavirus to be safely expressed on a lentiviral particle in order to assess its cell tropism. The system was developed and tested using the spike gene from CoV1087, a novel bat-borne coronavirus with a 70% similarity at both the coding RNA and amino acid level between its spike protein and that of porcine epidemic diarrhea virus strain CH-HNYF-14.
Overall this study resulted in the successful development of a lentiviral pseudotyping system which allows the efficient analysis of the cellular tropism of coronaviruses. Through the testing and development of this system, it was determined that CoV1087 could enter two human cell lines, 293T and CaCo-2 as well as one rabbit cell line, RK-13. This raises the possibility of the transfer of the virus to feral rabbits as well as the possibility of zoonotic human infections.
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Codran, Audrey. "Production de virus pseudotypes VSV/VHC : Etude de la fusion du VHC avec les cellules hôtes." Université Louis Pasteur (Strasbourg) (1971-2008), 2003. https://publication-theses.unistra.fr/public/theses_doctorat/2003/CODRAN_Audrey_2003.pdf.
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En raison de l'absence de système de culture cellulaire capable de propager efficacement le virus de l'hépatite C, peu de données sont disponibles concernant les phases précoces de l'infection. Afin d'étudier la fusion et la pénétration du VHC dans la cellule hôte, nous avons choisi de fabriquer des virus pseudotypes VSV/VHC destinés à mimer l'enveloppe du VHC dans les phases précoces de l'infection. Dans un premier temps, les glycoprotéines d'enveloppe du VHC (E1 et E2) sont modifiées pour être localisées à la membrane plasmique, site de bourgeonnement du VSV. Pour cela, les ectodomaines de E1 (jusqu'à l'acide aminé 311) et de E2 (jusqu'à l'acide aminé 661) sont fusionnés aux domaines transmembranaire et cytoplasmique de la glycoprotéine G du VSV. La protéine de fusion E1-TmG est également fusionnée à l'EGFP afin de faciliter sa détection. Les protéines de fusion EGFP-E1-TmG et E2-TmG sont produites dans la cellule grâce à deux adénovirus recombinants non réplicatifs. Pour produire les virus pseudotypes, les cellules sont tout d'abord infectées par ces deux adénovirus recombinants à 37 ʿC, puis par le VSVtsO45 à 40,5 ʿC, température à laquelle la glycoprotéine G mutée du VSV est retenue au niveau du réticulum endoplasmique. Les particules ainsi produites sont constituées d'une enveloppe contenant les glycoprotéines E1 et E2 modifiées et leur génome est celui du VSVtsO45. Par conséquent, l'infection de cellules par les pseudotypes à 33 ʿC (température permissive de la mutation thermosensible) permet la production de VSVtsO45. Dans un deuxième temps, nous nous intéressons aux mécanismes utilisés par le VHC pour pénétrer dans la cellule. Nous avons ainsi pu déterminer que la fusion des membranes virale et cellulaire se déroulait à pH acide et qu'elle avait lieu dans l'endosome. Finalement, nos résultats indiquent que les pseudotypes pénètreraient dans la cellule majoritairement par endocytose clathrine-dépendanteDue to the lack of cell culture system to propagate efficiently HCV, only few data are available concerning the early stages of HCV infection. In order to study HCV fusion and penetration into host cells, we have chosen to generate VSV/HCV pseudotyped viruses to mimic HCV envelope during the early stages of infection. First, E1 and E2 HCV glycoproteins are modified to be localized at the plasma membrane where VSV budding occurs. Thus, E1 (amino acid 311) and E2 (amino acid 661) ectodomains are fused to the transmembrane domain and the cytoplasmic tail of VSV G glycoprotein. Chimeric E1-TmG is also fused to EGFP to allow easy detection of this protein. Chimeric E1 and E2 are expressed in cells using non replicative recombinant adenoviruses. To produce pseudotypes, cells are first infected by both recombinant adenoviruses at 37 ʿC, and then super-infected by VSVtsO45 at 40. 5 ʿC. At this non-permissive temperature, the mutated VSV G glycoprotein is retained in the endoplasmic reticulum. Particles resulting from this triple infection are enveloped by modified E1 and E2 but their genome is still VSVtsO45. Therefore, infection with pseudotypes at 33 ʿC (permissive temperature for the mutation) will lead to production of VSVtsO45 virions. In a second step, we are interested in mechanisms involved in HCV penetration into the host cell. Pseudotypes are used to determine optimal pH required for cellular and viral membranes fusion. The use of such a tool has allowed to investigate and to understand part of the endocytic pathway implicated in early stages of HCV infection. Our results indicate that pseudotypes enter host cells via a pH-dependant route, that fusion of viral and cellular membranes occurs in the endosome, and that clathrin is involved in this mechanism
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Sandrin, Virginie. "Aspects fondamental et appliqué des pseudotypes rétroviraux : mécanismes d'assemblage et propriétés pour le transfert de gène." Lyon, École normale supérieure (sciences), 2005. http://www.theses.fr/2005ENSL0311.
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Gagnepain, Anaïs. "Évaluation de nouveaux pseudotypes de vecteurs lentiviraux pour le transfert de gènes dans les cellules hématopoiétiques." Thesis, Lyon, École normale supérieure, 2014. http://www.theses.fr/2014ENSL0939/document.
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Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souchesLentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols
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Ibrahim, Mazher Hassan. "History matching pressure response functions from production data." Texas A&M University, 2004. http://hdl.handle.net/1969.1/1486.
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This dissertation presents several new techniques for the analysis of the long-term production performance of tight gas wells. The main objectives of this work are to determine pressure response function for long-term production for a the slightly compressible liquid case, to determine the original gas in place (OGIP) during pseudosteady state (PSS), to determine OGIP in the transient period, and to determine the effects of these parameters on linear flow in gas wells.
Several methods are available in the industry to analyze the production performance of gas wells. One common method is superposition time. This method has the advantage of being able to analyze variable-rate and variable-pressure data, which is usually the nature of field data. However, this method has its shortcomings.
In this work, simulation and field cases illustrate the shortcomings of superposition. I present a new normalized pseudotime plotting function for use in the superposition method to smooth field data and more accurately calculate OGIP. The use of this normalized pseudotime is particularly important in the analysis of highly depleted reservoirs with large change in total compressibility where the superposition errors are largest.
The new tangent method presented here can calculate the OGIP with current reservoir properties for both constant rate and bottomhole flowing pressure (pwf) production. In this approach pressure-dependent permeability data can be integrated into a modified real gas pseudopressure,m(p), which linearizes the reservoir flow equations and provides correct values for permeability and skin factor. But if the customary real-gas pseudopressure, m(p) is used instead, erroneous values for permeability and skin factor will be calculated. This method uses an exponential equation form for permeability vs. pressure drop.
Simulation and field examples confirm that the new correction factor for the rate dependent problem improves the linear model for both PSS and transient period, whether plotted on square-root of time or superposition plots.
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Levy, Camille. "Nouveaux pseudotypes rétroviraux basés sur les glycoprotéines d'enveloppe de paramyxovirus : applications biothérapeutiques en thérapie génique et en vaccinologie." Thesis, Lyon, École normale supérieure, 2012. http://www.theses.fr/2012ENSL0709.
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Les paramyxovirus possèdent deux glycoprotéines d’enveloppe (gps): la protéine F, permettant la fusion avec la cellule hôte, et la protéine d’attachement appelée G, H ou HN. Les gps H et F d’une souche vaccinale du virus de la rougeole peuvent être incorporées sur des vecteurs lentiviraux (H/F-LV) permettant une transduction efficace des lymphocytes T et B humains non stimulés, habituellement réfractaires. Nous avons montré que les vecteurs H/F-LV sont capables de transduire des cellules B cancéreuses, activées et quiescentes, contrairement aux VSV-G-LV classiques. Leur utilisation in vivo est cependant confrontée à la présence d’anticorps neutralisants induits par la vaccination, dirigés majoritairement contre H. Après la mutation des 2 épitopes immunodominants de H, les vecteurs conservent leur tropisme et échappent à la neutralisation par les anticorps monoclonaux, mais sont toujours neutralisés par le sérum humain. Les souches émergentes de rougeole de génotype D, qui paraissent résister à la vaccination, présentent une glycosylation supplémentaire de la H. Introduite dans notre mutant, elle permet aux H/F-LV de transduire efficacement les cellules T et B en présence de sérum ou de sang total. Les pneumovirinae (le Virus Respiratoire Syncytial et le Métapneumovirus Humain (HMPV)) sont la première cause d’infections respiratoires chez le nourrisson, il n’existe pas de vaccin contre ces virus. Nous avons mis au point un système de Virus-Like Particle rétrovirales incorporant les gps F et G de HMPV (HMPV-VLPs). Injectées à des souris, les HMPV-VLP induisent une forte réponse d’anticorps neutralisants. De plus, suite à une épreuve virale, les souris sont protégées de l’infection par hMPVParamyxoviruses contain two envelope glycoproteins : the F protein allowing fusion with the host cell and an attachment protein, called G, H or HN. Lentiviral vectors pseudotyped with the Edmonston measles virus hemagglutinin and fusion glycoproteins (H/F-LVs) allowed for the first time efficient transduction of quiescent human T and B cells. We showed that H/F-LVs were also able to efficiently transduce quiescent and activated cancer B cells, in contrast to the classical VSV-G-LVs. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles inducing a humoral immune response exclusively directed against H. LVs pseudotyped with H-glycoproteins mutated in the 2 major epitopes escaped inactivation by monoclonal antibodies but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the mutated H/F-LVs allowing efficient transduction of quiescent lymphocytes in the presence of high concentration of MV antibody-positive human serum or total blood. Pneumovirinae (Respiratory Syncitial Virus and human metapnemovirus (HMPV)) are the leading cause of respiratory infections in infants and no vaccine is available against these viruses. We designed retroviral Virus-Like Particle incorporating HMPV F and G gps (HMPV-VLPs). HMPV-VLPs injected to mice induce a strong neutralizing antibody immune response in vivo. Furthermore, upon a viral challenge, HMPV-VLP vaccinated mice are protected against hMPV infection
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Islam, Tarin A. "Characterisation of axonal retrograde transport of rabies pseudotyped lentiviral vectors for application in gene therapy of motor neuron diseases." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/25142.
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Lentiviral vectors, such as those derived from Human Immunodeficiency Virus-1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV), can be targeted to the neurons by replacing their natural envelope with rabies-G glycoprotein (RV-G), through a process known as pseudotyping. They are retrogradedly transported to distal projecting neurons in vivo where transgene expression occurs, an approach with significant potential for gene therapy of motor neuron diseases. However, the molecular processes that underlie retrograde transduction are unchartered and barrier(s) which result in low transduction efficiencies are thus not defined. The project aimed to characterise the processes involved in the entry and endocytic trafficking of RV-G pseudotyped lentiviral vectors in a differentiated motor neuron cell line, NSC-34, and primary rat motor neurons. For the first time, the project demonstrates the co-internalisation of RV-G pseudotyped lentiviral vectors with its 3 specific receptors, namely p75 neurotrophin receptor (p75NTR), neural cell adhesion molecule (NCAM) and nicotinic acetylcholine receptor (nAchR). Furthermore, internalised lentiviral vectors follow a sequential Rab5 to Rab7 endosomal maturation along the axonal endocytic pathway. Using the process of tetracysteine tagging, a small genetic tag was successfully introduced into the matrix of the vector capsid and labelling of the tag with the biarsenical dye FlAsH was optimised. Utilising these and other differently labelled vectors, we demonstrate through live cell imaging, that it is the RV-G pseudotype that confers on the vectors the ability to traffic retrogradedly. This trafficking pathway utilises non-acidic axonal carriers as previously demonstrated for the p75NTR and tetanus toxin trafficking in motor neurons. Additionally, the nAchR is also targeted to this retrograde trafficking pathway. Furthermore retrograde transport was studied in compartmented microfluidic motor neuron cultures and this trafficking was found to be both rapid and efficient. This suggests that the barrier(s) to efficient transduction with these vectors is post axonal transport. Additionally, studies to isolate and characterise axonal retrograde endosomes in primary motor neurons based on a system of pull-down of magnetic nano particles (MNPs) bound to a streptavidin displaying lentiviral vector were performed. The project optimised streptavidin expressing vector production and shown their ability to bind biotin coated magnetic nano particles (MNPs) and transduce motor neurons with increased efficiency. Magnetic pull-down of the vector containing endosomes and subsequent proteomics have revealed a number of potential trafficking partners of RV-G pseudotyped lentiviral vectors. Taken together, this thesis characterises the processes involved in the entry and endocytic trafficking of RV-G pseudotyped lentiviral vectors and identifies the barrier to efficient transduction of motor neurons as post axonal transport and thereby opening up avenues to improve gene therapy vectors.
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Gollan, Timothy J. "Altering the Tropism of Retroviral Vectors For In Vivo Gene Therapy: Pseudotyped Virus Targeting by Ligand-Receptor Interactions: A Dissertation." eScholarship@UMMS, 2002. http://escholarship.umassmed.edu/gsbs_diss/226.
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A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope.
These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.
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Jäkel, Melanie [Verfasser], and Gerd [Akademischer Betreuer] Sutter. "In vivo characterization of a pseudotyped vesicular stomatitis virus for the treatment of Hepatocellular carcinoma / Melanie Jäkel ; Betreuer: Gerd Sutter." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1206096616/34.
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Le, Goff Jérôme. "Interactions entre le VIH-1 et l' herpes simplex de type 2 dans le microenvironnement cellulaire et génital." Paris 7, 2005. http://www.theses.fr/2005PA077105.
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Coquin, Youna. "Caractérisation de vecteurs lentiviraux pseudotypés par les syncytines murines et de leurs cibles cellulaires in vitro et in vivo." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLE039.
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Les vecteurs lentiviraux (LV) de thérapie génique sont des particules recombinantes qu'il est possible de pseudotyper avec diverses glycoprotéines d'enveloppe afin de modifier l'adressage cellulaire ou leurs propriétés immunogéniques. Mon travail de thèse a exploré l'hypothèse que la VSVG, la glycoprotéine d'enveloppe la plus utilisée pour pseudotyper les LV, puisse être remplacée par des protéines cellulaires afin d'obtenir des particules bien tolérées et administrables in vivo. J'ai utilisé les syncytines murines, qui sont des glycoprotéines d'origine rétrovirale endogène et qui ont des propriétés fusogéniques et un potentiel immunosuppressif. Mes résultats ont montré la possibilité de produire des LV pseudotypés par les syncytines murines A et B. Les particules ainsi produites (LV-SynA et LV-SynB) sont infectieuses en présence d'un adjuvant de transduction et présentent une forte capacité à transduire les lymphocytes B murins in vitro. Les vecteurs LV-Syn sont capables de transduire des lymphocytes B quiescents, ainsi que des cellules précurseurs des lymphocytes B issus de moelle osseuse de souris. L'administration de LV-SynA in vivo chez la souris par voie intraveineuse permet d'observer un transfert de gène stable. Un signal est observé dans la rate et plus particulièrement dans les lymphocytes B au niveau des centres germinatifs, en accord avec les résultats obtenus in vitro. Mes travaux confirment donc, et étendent, les observations préalables du laboratoire sur la transduction de lymphocytes B naïfs humains avec les syncytines humaines, et suggèrent un fort tropisme des syncytines en général pour les lymphocytes B. Par ailleurs, mes résultats montrent qu'in vivo, le poumon est également ciblé par les LV-SynA, en accord avec l'expression récemment identifiée du récepteur à la syncytine A, Ly6e, dans cet organe. Il semble que le transfert de gène puisse être obtenu dans l'épithélium alvéolaire et il est possible de transduire des cellules primaires épithéliales pulmonaires humaines avec les LV-Syn in vitro. Globalement, mes résultats montrent une nouvelle perspective d'interaction entre les syncytines et le système immunitaire à travers la transduction des lymphocytes B. Mes résultats permettent également d'envisager de nouvelles perspectives pour le transfert de gène dans le poumon in vivo et pour développer de nouvelles approches de thérapie génique par voie systémiqueLentiviral gene transfer vectors (LV) used in gene therapy are recombinant virus-like particles that can be pseudotyped with diverse envelope glycoproteins in order to modify their cellular targeting or their immunogenic properties. My thesis work explored the hypothesis that VSVG, the most-commonly-used viral envelope glycoproteins of LV, could by replaced by cellular glycoproteins to obtain well-tolerated particles that can be administered in vivo. I used murine syncytins which are glycoproteins of endogenous retroviral origin and which have fusogenic properties and immunosuppressive potential. My results showed the possibility of producing LV pseudotyped with murine syncytins A or B. The LV-SynA or LV-SynB particles produced were infectious in the presence of a transduction adjuvant and efficiently transduced murine B cells in vitro, including quiescent B cells and bone marrow B precursor cells. In vivo LV-SynA intravenous administration in mice enabled stable gene transfer. A positive signal was observed in the spleen, and especially in B cells at the level of the germinal centers, confirming in vitro observations. These results confirm and extend previous observations from the laboratory showing that human naive B cells can be transduced by LV pseudotyped with human syncytins, and emphasize the marked interactions between syncytins and B cells. Moreover, my results showed that in vivo in mice LV-SynA targets the lungs, possibly epithelial alveolar cells, in line with the recent findings of Syncytin A receptor Ly6e expression in the lung. Gene transfer was also obtained in human primary pulmonary epithelial cells in vitro with LV-Syn. Overall, my results reveal new interactions between syncytins and the immune system through the transduction of B cells. My results also provide novel perspectives for gene delivery in vivo in the lung and for designing new systemic gene therapy approaches
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Molina, Rosa-Maria. "Facteurs influençant l'expression de vecteurs rétroviraux dérivés des virus ALSV in vitro : rôle des pseudotypes rétroviraux sur la spécificité tissulaire du transfert de gènes intraembryonnaire chez les oiseaux." Lyon 1, 1994. http://www.theses.fr/1994LYO10097.
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Les animaux transgeniques constituent actuellement un modele particulierement adapte pour de nombreuses etudes in vivo tant en recherche fondamentale qu'en recherche appliquee. Les vecteurs mis au point dans notre laboratoire pour transferer des genes exogenes chez les oiseaux sont derives des retrovirus aviaires alsv et produits en condition helper-free. Ils infectent les cellules permissives et l'information genetique qu'ils vehiculent s'integre efficacement dans le genome cellulaire. Cependant, l'expression stable et efficace des genes transferes depend de multiples facteurs. Ce travail a montre que les sequences regulatrices de la transcription et de la traduction originaires des virus rav-1 et rav-2 autorisent une expression stable et efficace du gene marqueur lacz, introduit par infection retrovirale dans la lignee cellulaire qt6. De plus, la nature et le niveau de la selection appliquee sur les cellules qt6 infectees revelent une grande variabilite de l'efficacite et la stabilite d'expression du gene marqueur transfere. Par ailleurs, l'emploi de stocks viraux portant un genome vecteur identique mais des enveloppes appartenant a des sous-groupes viraux differents, et de titre eleve, permet de cibler l'infection de certains organes in vivo. Cependant, le tropisme d'expression du gene lacz varie egalement en fonction du stade embryonnaire au cours duquel est effectuee l'infection retrovirale. De plus, certains tissus, comme le foie et l'estomac, expriment rarement le gene marqueur bien qu'ils aient incorpore l'information genetique portee par les vecteurs retroviraux
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Lim, Chee Yee. "Understanding transcriptional regulation through computational analysis of single-cell transcriptomics." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267786.
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Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.
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Waern, Johan Martin [Verfasser], and Michael [Akademischer Betreuer] Ott. "Cell-specific infection of human cells mediated by lentiviral vectors pseudotyped with measles virus hemagglutinin fused to single chain antibodies / Johan Martin Waern ; Akademischer Betreuer: Michael Ott ; Klinik für Gastroenterologie, Hepatologie und Endokrinologie der Medizinischen Hochschule Hannover." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2018. http://d-nb.info/1151400343/34.
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Waern, Johan [Verfasser], and Michael [Akademischer Betreuer] Ott. "Cell-specific infection of human cells mediated by lentiviral vectors pseudotyped with measles virus hemagglutinin fused to single chain antibodies / Johan Martin Waern ; Akademischer Betreuer: Michael Ott ; Klinik für Gastroenterologie, Hepatologie und Endokrinologie der Medizinischen Hochschule Hannover." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2018. http://nbn-resolving.de/urn:nbn:de:gbv:354-2017111580.
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Pagliaro, Sarah Beatriz De Oliveira. "Transcriptional control induced by bcr-abl and its role in leukemic stem cell heterogeneity. Single-Cell Transcriptome in Chronic Myeloid Leukemia: Pseudotime Analysis Reveals Evidence of Embryonic and Transitional Stem Cell States Single Cell Transcriptome in Chronic Myeloid Leukemia (CML): Pseudotime Analysis Reveals a Rare Population with Embryonic Stem Cell Features and Druggable Intricated Transitional Stem Cell States A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC) Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML)." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASQ032.
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La leucémie myéloïde chronique est une hématopoïèse maligne clonale, caractérisée par l'acquisition de la translocation t (9;22) conduisant au chromosome Ph1 et à son homologue l'oncogène BCR-ABL, dans une cellule souche hématopoïétique très primitive. La LMC est un modèle de thérapies ciblées, car il a été démontré que la preuve de la faisabilité du ciblage de l'activité tyrosine kinase (TK) BCR-ABL à l'aide d'inhibiteurs de TK (TKI) entraîne des réponses et des rémissions majeures. Cependant, les problèmes actuels rencontrés dans ces thérapies sont la résistance des cellules souches leucémiques primitives et leur persistance qui serait liée à l'hétérogénéité des cellules souches au moment du diagnostic, ce qui conduit à la sélection clonale de cellules résistant aux thérapies TKI. J'ai appliqué la technologie de l'analyse du transcriptome des cellule uniques aux cellules de la LMC en utilisant un panel de gènes impliqués dans différentes voies, combinée à l'analyse d'inférence de trajectoire au modèle d'expression des gènes. Les résultats ont montré un état transitoire des cellules souches comprenant des gènes embryonnaires identifiés dans les cellules de la LMC au moment du diagnostic, ce qui pourrait contribuer à la résistance et à la persistance de la LSC. En outre, l'oncoprotéine Bcr-Abl est la tyrosine kinase constitutivement active produite par le gène chimérique BCR-ABL dans la leucémie myéloïde chronique (LMC). Les cibles transcriptionnelles de Bcr-Abl dans les cellules leucémiques n'ont pas été étudiées de manière approfondie. Une expérience de transcriptome utilisant la lignée cellulaire UT7 hématopoïétique exprimant BCR-ABL, a identifié la surexpression du facteur d'élongation eucaryote kinase 2 (eEF2K) qui joue un rôle majeur dans la survie des cellules en cas de privation de nutriments. Dans l'ensemble, les données suggèrent que la surexpression de eEF2K dans la LMC est associée à une sensibilité accrue à la privation de nutrimentsChronic myeloid leukemia is a clonal hematopoietic malignancy, characterized by the acquisition of the t (9;22) translocation leading to Ph1 chromosome and its counterpart BCR-ABL oncogene, in a very primitive hematopoietic stem cell. CML is a model of targeted therapies as the proof of concept of the feasibility of targeting the tyrosine kinase (TK) activity BCR-ABL using TK inhibitors (TKI) has been shown to lead to major responses and remissions. However, the current problems encountered in these therapies are primitive leukemic stem cells resistance and their persistence which is thought to be related to the heterogeneity of the stem cells at diagnosis leading to clonal selection of cells resisting to TKI therapies. I have applied the technology of single cell transcriptome analysis to CML cells using a panel of genes involved in different pathways combined with trajectory inference analysis to the gene expression pattern. The results showed a transitional stem cell states including embryonic genes identified in CML cells at diagnosis which could contribute to LSC resistance and persistence. Furthermore, the oncoprotein Bcr-Abl is the constitutively active tyrosine kinase produced by the chimeric BCR-ABL gene in chronic myeloid leukemia (CML). The transcriptional targets of Bcr-Abl in leukemic cells have not been extensively studied. A transcriptome experiment using the hematopoietic UT7 cell line expressing BCR-ABL, has identified the overexpression of eukaryotic elongation factor kinase 2 (eEF2K) which plays a major role in the survival of cells upon nutrient deprivation. Overall, the data suggest that overexpression of eEF2K in CML is associated with an increased sensitivity to nutrient-deprivation
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Lin, Po-Yin, and 林柏吟. "Expressing dengue virus 2 E protein for pseudotype formation of murine leukemia virus with dengue virus envelope." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/35241300985270384102.
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碩士國立交通大學
生化工程研究所
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Dengue virus (DV), a member of the family Flaviviridae, is an enveloped, single-stranded positive sense RNA virus. Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by the dengue viruses that represent a global public health problem. In the process of virus entry, the envelope protein (E protein) plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. The extracellular domain of E protein is thought to interact with the host cell receptor. There is no discernable change in the host cell that infected by dengue virus. In general, one can only use plaque assay to determine whether the propagation is successful. However, not every cell line infected can produce plaques and even if it can, the plaque formation needs 5~7 days. Those events make research in dengue virus difficult. The objectives of this study were to generate infectious pseudotyped-particles that were assembled by functional dengue virus E proteins onto retroviral core particles. In this assay, mammalian cells are cotransfected with 3 plasmids, one expressing the retroviral proteins—expect the envelope—from murine leukemia virus, another expressing the replication-defective retroviral genome with β-galactosidase gene marker, and the other expressing the E gene from dengue virus. In conclusion: First, the recombinant proteins of DV-2 E protein fused with His-tag can be detected in the E.coli expression system by Western blotting analysis but not in BHK21. Second, the DV E proteins can reach the cell surface because the stable cell lines transfected with the plasmid expressing DV E gene displayed syncytium. Third, in this study, no pseudotype viruses were detected. Further studies are required.
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Nogueira, Rodrigo José Gameiro. "Pseudotyped lentiviral vectors for gene therapy: impact of envelope glycoproteins." Master's thesis, 2019. http://hdl.handle.net/10362/89669.
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The potential of gene therapy to treat a broad range of diseases, ranging from cancer to monogenic diseases, makes it a powerful approach to address medical needs that are currently unmet by conventional medicine. Lentiviral vectors have been the viral vectors experiencing the highest growth in gene therapy clinical trials due to their large genetic payload, the ability to transduce both dividing and non-dividing cells, permanently integrating into the target cell genome and increased safety when compared to Gammaretrovirus. However, the utilization of lentiviral vectors in gene therapy has been conditioned by the currently available production systems. The development of constitutive producer cell lines will provide a better alternative to cope with the increasing necessity of large-scale production of lentiviral vectors.
This work focused on the evaluation of GaLV10A1ΔR envelope glycoprotein potential for stable expression, in order to enable its use in constitutive producer cells. This was based on a previously developed cell line, HEK 293T SLC20A1 knock-out, especially designed to cope with the cytotoxicity of GaLV10A1ΔR. To achieve stable expression of GaLV envelope glycoproteins new plasmids were constructed. An analytical method for syncytia quantification, based on cell size distribution, was also successfully developed. This work showed that SLC20A1 deletion enabled GaLV10A1ΔR expression, eliminating the cytotoxicity associated with its expression. More importantly, SLC20A1 knock-out enabled the establishment of a cell population stably expressing GaLV10A1ΔR. SLC20A1 deletion led to a decrease in cell growth, however it was restored by SLC20A2 complementation. Altogether, this thesis supports the viral receptor knock-out strategy to develop new cell lines to express glycoproteins with fusogenicity-derived cytotoxicity and provides new insights that will help improve this strategy.
This work contributes to the state-of-the-art of the lentiviral vector-based gene therapy, providing new insights that will improve the development of constitutive producer cells in the near future.O potencial da terapia génica para tratar um largo espetro de doenças, desde o cancro até monogénicas, torna-a uma abordagem poderosa para colmatar necessidades médicas que atualmente não são atendidas pela medicina convencional. Os vetores lentivirais foram os vetores virais que apresentaram o maior crescimento em ensaios clínicos de terapia génica. Isto deve-se à sua grande carga genética, à capacidade de transduzir células em divisão e não-divisão, integração permanente no genoma da célula hospedeira e maior segurança comparativamente aos Gammaretrovirus. No entanto, a sua utilização em terapia génica é condicionada pelos sistemas de produção de vetores lentivirais atualmente disponíveis. O desenvolvimento de linhas celulares produtoras constitutivas será uma melhor alternativa para lidar com a necessidade crescente de sistema de produção em larga escala de vetores lentivirais. Este trabalho avaliou a potencial utilização da glicoproteína GaLV10A1ΔR em expressão estável, com o objetivo de permitir a sua utilização no estabelecimento de linhas celulares produtoras constitutivas. Este trabalho baseou-se numa linha celular já desenvolvida, HEK 293T SLC20A1 KO, especialmente projetada para lidar com a citotoxicidade de GaLV10A1ΔR. Aqui, construímos novos plasmídeos capazes de expressar glicoproteínas estavelmente. Desenvolvemos também um método analítico de quantificação de sincícios. Este trabalho mostrou que é possível eliminar a citotoxicidade associada com GaLV10A1ΔR através da deleção de SLC20A1. Mostrámos também que a deleção de SLC20A1 permitiu a expressão estável de GaLV10A1ΔR. A deleção desta proteína levou à diminuição do crescimento HEK 293T SLC20A1 KO, no entanto, este foi recuperado com a sobreexpressão de SLC20A2. Em conclusão, este trabalho suporta a estratégia baseada na deleção de recetores virais para a expressão estável de glicoproteinas citotóxicas. Esta tese contribui para o estado da arte da terapia génica baseada em vetores lentivirais, fornecendo novo conhecimento que permitirá melhorar o desenvolvimento de linhas celulares produtoras constitutivas num futuro próximo.
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Núñez, Jisette González. "NON-PATHOGENIC VIRAL VECTORS PSEUDOTYPED WITH THE SARS-COV-2 S PROTEIN TO STUDY DRUG REPURPOSING AND IMMUNIZATION STRATEGIES." Master's thesis, 2021. http://hdl.handle.net/10316/99147.
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Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de FarmáciaO coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) é o vírus responsável pela doença coronavírus 2019 (COVID-19). Os primeiros casos de SARS-CoV-2 foram reportados na China e, em setembro de 2021, mais de 228 milhões de pessoas foram infectadas e mais de 4 milhões de mortes foram registadas por esta doença. Embora a origem do SARS-CoV-2 ainda seja desconhecida, a explicação mais aceite é um evento zoonótico. O SARS-CoV-2 pertence à família Coronaviridae (ordem Nidovirales), um grupo de vírus de (+) RNA de cadeia única, com envelopes que podem infectar três classes principais de vertebrados: mamíferos, pássaros e peixes. Todos os membros desta família compartilham um grupo de características, tais como a presença de uma nucleocápside (N) de múltiplas cópias de uma fosfoproteína, a proteína de membrana integral (M) e a proteína N-glicosilada spike (S). O principal mecanismo de infecção celular do SARS-CoV-2 baseia-se na afinidade da proteína S pelo receptor celular da enzima conversora de angiotensina 2 (ACE2).No presente trabalho foram investigados 1970 fármacos aprovados pela FDA pela sua capacidade de inibir ou potenciar a infecção de células HEK-293T, expressando o receptor celular ACE2 humano, por vectores lentivirais pseudotipados de forma a exibir a proteína S do SARS-CoV-2 à sua superfície e codificando a proteína luciferase. O tratamento foi adicionado às células concomitante com os vetores lentivirais pseudotipados, e posteriormente, a capacidade de infecção foi quantificada pela intensidade de luminescência emitida pelas células.Também foi avaliada a capacidade de imunização desta partícula em murganhos C57BL/6, para tal, vetores lentivirais pseudotipados com a proteínas S foram administrados em murganhos fêmeas por via intraperitoneal e um reforço adicional por via intranasal.Além disso, empregamos as mesmas partículas lentivirais pseudotipadas de proteína S para estudar sua capacidade de imunização em camundongos C57BL/6 para esse fim, LVP foi administrado a camundongos fêmeas por via intraperitoneal e reforço adicional por via intranasal.Foram identificados 66 fármacos que inibiram a infecção da partícula pseudotipada, sendo 33 deles relatados pela primeira vez neste trabalho e 59 fármacos que mostraram um aumento da infecção nas células testadas. No total, 124 fármacos estão a ser avaliados por curvas dose-resposta sendo que dos 25 fármacos já avaliados, apenas oito apresentaram correlação dose-resposta.Além disso, a estratégia de imunização aqui desenvolvida com a partícula pseudotipada induziu a produção de anticorpos IgG contra a proteína spike nos murganhos imunizados. A análise do soro destes animais também revelou a capacidade de inibir a infecção da partícula pseudotipada em células HEK-hACE2, demonstrando a viabilidade e eficácia dos anticorpos IgG produzidos. Por fim, estes resultados contribuem para a geração de novas estratégias de tratamento e imunização para a COVID-19 e evidenciam a necessidade de continuar este estudo para compreender as novas descobertas aqui apresentadas.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (COVID-19). SARS-CoV-2 outbreak began in China, and as of September of 2021, more than 228 million people have been infected and more than 4 million deaths were registered. Although the origin of SARS-CoV-2 is still unknown, the most parsimonious explanation is a zoonotic event. SARS-CoV-2 belongs to the Coronaviridae family (order Nidovirales), a group of enveloped and positive single-strand RNA viruses that can infect three major classes of vertebrates: mammals, birds, and fish. All members of this family share a group of characteristics such as the presence of a nucleocapsid (N) of multiple copies of a phosphoprotein, the integral membrane protein (M) and the N-glycosylated spike protein (S). The major cell infection mechanism of SARS-CoV-2 is based on the affinity of the S protein by the cell receptor angiotensin- converting enzyme- 2 (ACE2).In the present work, we investigated the capacity of a library of 1970 FDA-approved drugs to inhibit or potentiate infection of HEK-293T cells overexpressing the human ACE2 cellular receptor by a pseudotyped lentiviral particle exhibiting the S protein of SARS-CoV-2 at its surface and encoding luciferase. The drug treatment and pseudotyped particles were added to the cells at the same time and after an incubation period, the infection capacity was measured as luminescence emission by the cells.Furthermore, we employed the same S-protein pseudotyped lentiviral particles to study its immunization capacity in C57BL/6 mice for that purpose, LVP were administered to female mice via intraperitoneal route and further boost through intranasal route.We have identified 66 drugs that inhibit infection of the pseudotyped LVP, being 33 drugs first reported in this work. Furthermore, we also identified 59 drugs that potentiated SARS-CoV-2 pseudotyped LVP infection. Dose-response curves of the identified compounds are still being analyzed and from the 25 drugs already evaluated, eight showed a dose-response correlation.Finally, the immunization strategy here developed with the LVP-Spike induced the production of IgG antibodies against S protein in the immunized mice. The analysis of immunized animal serum also showed the capacity to inhibit the infection of the LVP-Spike in HEK-hACE2 cells, demonstrating the viability and efficacy of the produced IgG antibodies. In conclusion, these results contribute to the generation of new strategies for COVID-19 treatment and immunization, and evidence the need for continuation of this study to better explore and develop the new finds here presented.
Outro - FCT project - 587-ViraVector4Covid - Viral Vectors for discovery of Covid19 pathogenesis, treatment and vaccination
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Drăgan, Anette [Verfasser]. "Entwicklung klinisch charakterisierter HCV-Pseudotypen zur Untersuchung der HCV-assoziierten Kryoglobulinämie und der Virusneutralisierung durch Antikörper / vorgelegt von Anette Drăgan." 2010. http://d-nb.info/1006604464/34.
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Wu, Chia Jen, and 吳嘉仁. "The establishment of pseudotyped adeno-associated virus 2/9-delivered CCL11 shRNA and CC10 gene to alleviate lung inflammation in allergen-sensitized murine model." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/56930458481372748776.
Full textAbstract:
博士長庚大學
生物醫學研究所
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Asthma is a chronic airway inflammatory disease characterized by eosinophilic infiltration and airway hyperresponsiveness. The over-activated Th2 and lung epithelial cells express many different cytokines and chemokines that mainly contribute to the severity of lung inflammation. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as a major chemokine for eosinophil recruitment. Clara cell 10 kDa protein (CC10) that is highly expressed and secreted from airway epithelial cells and exhibits anti-inflammatory and immunomodulatory effects. Pseudotyped adeno-associated virus (AAV) 2/9 vector, composed of AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether AAV2/9 vector virus carrying the small hairpin RNA targeting CCL11 (expressed by dual CMV/U6 promoter), and carrying CC10 cDNA could reduce eosinophilia, lung inflammation, and Th2 responses in allergen-induced asthma mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 protein (rDp2) or ovalbumin (OVA). AAV2/9 vector viruses were intratracheally injected three days before the first challenge. In shRNA delivery experiments, the data showed that AAV2/9 sh47 vector virus significantly reduced airway hyperresponsiveness (AHR), airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukin (IL)-4, IL-5, IL-10, and IL-13, were significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 vector virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. Lung tissue remodeling, including collagen deposition and goblet cell hyperplasia was alleviated. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. In CC10 cDNA delivery experiments, the results showed that AAV2/9 CC10 vector virus significantly reduced AHR, the accumulation of CCL11, IL-4, IL-5, IL-6, and IL-13 levels, and eosinophilia in the lungs of sensitized mice. CC10 level in OVA-sensitized mice was rescued with the administration of AAV2/9 CC10 vector virus. Lung tissue remodeling was relieved as in the CCL11 sh47 experiments. The systemic OVA-specific IgG1 and IgE, or Th2 cytokines from OVA stimulated splenocytes were not affected. The results demonstrate that AAV2/9 vector virus relieved local instead of systemic inflammatory responses. Therefore, AAV2/9 shRNA vector virus with CMV/U6 promoter specific to CCL11 efficiently down-regulates eosinophilia, and AAV2/9 CC10 vector virus to ensure sufficient CC10 expression and anti-inflammatory effect in asthmatic mice might be applied as a novel therapeutic approach for asthma.