Publicação
Novel fluorescent cell-based sensors for detection of viral pathogens
| Resumo: | Viruses are major pathogenic agents that can cause a variety of serious diseases. Indeed, the establishment of cell culture techniques and recombinant virus manipulation contributed for the development of viral-based biotherapies, like gene therapy or vaccines, which require accurate and fast quantification of virus. Despite the numerous titration methods existing nowadays, the majority of them are not able to provide a robust and fast quantification, essential for their clinical application. Moreover, most of them provide indirect measurements of infectious particles, over-estimating virus infectivity, and some rely on virus modification, e.g. by making use of reporter genes (labelled-viruses), which are not allowed when using those virus for clinical applications. As so, the development of a new system capable to cope these drawbacks is of paramount importance for research, diagnostics and industry. In this work, genetically encoded switch-on fluorescent mammalian cell-based assays for detection and quantification of label-free Adenoviruses, using the adenoviral protease (Adenain) as a trigger of the sensor, were developed. Three different main strategies were designed based on structural distortion of a fluorescent protein (GFP – Green Fluorescent Protein), preventing fluorescence emission: GFP VISENSOR (cGFP), Embedded Split-GFP VISENSOR (eS11) and Circular Split-GFP VISENSOR (cS11). Upon Adenain proteolytic processing, structural distortion is relieved and fluorescence emission is reconstituted. VISENSORS performance was assessed by optimizing the best combination of backbone structure and cleavage site, initially by a transient screening and later confirmed on a more biological context, where cells stably expressing the sensor were infected by human Adenovirus serotype 5. Despite eS11 and cGFP displaying similar signal to noise ratio (SNR) performances, cS11 strategy seems the most promising, reaching a signal to noise ratio of 3.12 at 72 hours post-infection. Virus detection was accomplished as soon as 24 hours post-infection in all strategies. Moreover, this work validated the use of VISENSORS as an Adenain dependent sensor and specific for Adenovirus. An attempt to reach maximum distortion and improving SNR performances, a parallel strategy was implemented by structurally distorting both split-GFP fragments. However, the results were not promising. A detailed characterization of the best strategy will be performed as future work, using cell clones stably expressing the sensor to assess VISENSORS’ applicability to Adenovirus quantification. VISENSORS show great potential to deliver a fast, reliable and accurate method for virus and viral vector detection and quantification, much needed not only in the development of viral based-biotherapies, but also for diagnostic and clinical applications. |
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| Autores principais: | Freitas, Daniela Filipa da Cruz |
| Assunto: | Deteção e quantificação de vírus Adenovírus Vírus sem marcação Biossensores Teses de mestrado - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Viruses are major pathogenic agents that can cause a variety of serious diseases. Indeed, the establishment of cell culture techniques and recombinant virus manipulation contributed for the development of viral-based biotherapies, like gene therapy or vaccines, which require accurate and fast quantification of virus. Despite the numerous titration methods existing nowadays, the majority of them are not able to provide a robust and fast quantification, essential for their clinical application. Moreover, most of them provide indirect measurements of infectious particles, over-estimating virus infectivity, and some rely on virus modification, e.g. by making use of reporter genes (labelled-viruses), which are not allowed when using those virus for clinical applications. As so, the development of a new system capable to cope these drawbacks is of paramount importance for research, diagnostics and industry. In this work, genetically encoded switch-on fluorescent mammalian cell-based assays for detection and quantification of label-free Adenoviruses, using the adenoviral protease (Adenain) as a trigger of the sensor, were developed. Three different main strategies were designed based on structural distortion of a fluorescent protein (GFP – Green Fluorescent Protein), preventing fluorescence emission: GFP VISENSOR (cGFP), Embedded Split-GFP VISENSOR (eS11) and Circular Split-GFP VISENSOR (cS11). Upon Adenain proteolytic processing, structural distortion is relieved and fluorescence emission is reconstituted. VISENSORS performance was assessed by optimizing the best combination of backbone structure and cleavage site, initially by a transient screening and later confirmed on a more biological context, where cells stably expressing the sensor were infected by human Adenovirus serotype 5. Despite eS11 and cGFP displaying similar signal to noise ratio (SNR) performances, cS11 strategy seems the most promising, reaching a signal to noise ratio of 3.12 at 72 hours post-infection. Virus detection was accomplished as soon as 24 hours post-infection in all strategies. Moreover, this work validated the use of VISENSORS as an Adenain dependent sensor and specific for Adenovirus. An attempt to reach maximum distortion and improving SNR performances, a parallel strategy was implemented by structurally distorting both split-GFP fragments. However, the results were not promising. A detailed characterization of the best strategy will be performed as future work, using cell clones stably expressing the sensor to assess VISENSORS’ applicability to Adenovirus quantification. VISENSORS show great potential to deliver a fast, reliable and accurate method for virus and viral vector detection and quantification, much needed not only in the development of viral based-biotherapies, but also for diagnostic and clinical applications. |
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