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Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools
Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccinati...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2023
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| _version_ | 1867614094064877568 |
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| access_status_str | Open Access |
| author | Isaacs, Abdul |
| author2 | Rybicki, Edward |
| author_browse | Isaacs, Abdul Rybicki, Edward |
| author_facet | Rybicki, Edward Isaacs, Abdul |
| author_sort | Isaacs, Abdul |
| collection | Thesis |
| description | Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccination offers a solution to reduce the difficulties that accompany viral outbreaks. Lujo virus, an emerging arenavirus responsible for causing a devastating haemorrhagic fever with an 80% mortality rate, currently has no vaccines or diagnostic tools available. In this study we developed a production pipeline in HEK293T cells and N. benthamiana for the protein LUJV GP-C∆TM (Lujo virus glycoprotein precursor without the transmembrane region). The LUJV GP-C∆TM was constructed from gene sequences of the envelope glycoprotein of Lujo virus and then adapted to production in HEK293T cells using the DNA expression vector pTHpCapR. An N. benthamiana plant protein expression system was developed in parallel to compare the production utility of both systems with an emphasis being placed on the plant system. Plant expression systems are arguably cheaper and more easily automatable than traditional mammalian expression technologies. This makes them suitable for vaccine protein production in lower socio-economic countries that have an overwhelming burden of disease and poor health care systems. LUJV GP-C∆TM was successfully expressed in both HEK293T cells and N. benthamiana. It was confirmed that the LUJV GP-C∆TM undergoes the post translational modifications glycosylation and proteolytic cleavage in recombinant HEK293T cells and was produced in a conformation that allowed successful purification via a His tag sequence that was inserted into the LUJV GP-C∆TM protein gene sequence. On the other hand, N. benthamiana does not endogenously express the Site-1 protease needed for cleavage of the Lujo glycoprotein, and thus this needed to be co-expressed. Attempts were made to detect the Site-1 protease including extraction into buffers with different pHs and ammonium sulfate precipitation to concentrate the protein. However, I was not able elicit proteolytic cleavage of the LUJV GP-C∆TM nor detect the Site-1 protease in N. benthamiana. This is probably attributable to the innate responses against the hostile nature of proteases to non-native expression hosts. Proteins were expressed in N. benthamiana through the use of previously established protocols utilizing recombinant Agrobacterium tumefaciens to deliver synthesized genes to plant cells and induce their expression. It was determined that co-expression of the molecular folding chaperone calreticulin is necessary for LUJV GP-C∆TM to accumulate at detectable levels. Co-expression of an oligossacharyl transferase LmSTT3D, isolated from Leishmania major, may increase the glycan occupancy of the LUJV GP-C∆TM protein, indicated by a molecular mass shift. However, further experimental lines of evidence are needed -such as glycosylation mapping to determine if this is the case. Other parameters such as the optimal day of protein harvest and optical density of recombinant Agrobacterium tumefaciens strains were recorded. Collectively these findings serve as a prototype pipeline for the production of commercially relevant immunogens, diagnostic tools and virus-like particles. This study narrows the focus for bottlenecks in plant protein production, not just for Lujo virus proteins, but for arenaviruses generally and potentially also other haemorrhagic fever-causing viruses. Future efforts should be directed towards addressing the barriers to plant production of complex viral antigens, and to further investigate the utility of mammalian cell-produced LUJV GPC∆TM in animal studies. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/38017 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:46:34.468Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/38017 Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools Isaacs, Abdul Rybicki, Edward Meyers Ann Elizabeth Molecular and Cell Biology Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccination offers a solution to reduce the difficulties that accompany viral outbreaks. Lujo virus, an emerging arenavirus responsible for causing a devastating haemorrhagic fever with an 80% mortality rate, currently has no vaccines or diagnostic tools available. In this study we developed a production pipeline in HEK293T cells and N. benthamiana for the protein LUJV GP-C∆TM (Lujo virus glycoprotein precursor without the transmembrane region). The LUJV GP-C∆TM was constructed from gene sequences of the envelope glycoprotein of Lujo virus and then adapted to production in HEK293T cells using the DNA expression vector pTHpCapR. An N. benthamiana plant protein expression system was developed in parallel to compare the production utility of both systems with an emphasis being placed on the plant system. Plant expression systems are arguably cheaper and more easily automatable than traditional mammalian expression technologies. This makes them suitable for vaccine protein production in lower socio-economic countries that have an overwhelming burden of disease and poor health care systems. LUJV GP-C∆TM was successfully expressed in both HEK293T cells and N. benthamiana. It was confirmed that the LUJV GP-C∆TM undergoes the post translational modifications glycosylation and proteolytic cleavage in recombinant HEK293T cells and was produced in a conformation that allowed successful purification via a His tag sequence that was inserted into the LUJV GP-C∆TM protein gene sequence. On the other hand, N. benthamiana does not endogenously express the Site-1 protease needed for cleavage of the Lujo glycoprotein, and thus this needed to be co-expressed. Attempts were made to detect the Site-1 protease including extraction into buffers with different pHs and ammonium sulfate precipitation to concentrate the protein. However, I was not able elicit proteolytic cleavage of the LUJV GP-C∆TM nor detect the Site-1 protease in N. benthamiana. This is probably attributable to the innate responses against the hostile nature of proteases to non-native expression hosts. Proteins were expressed in N. benthamiana through the use of previously established protocols utilizing recombinant Agrobacterium tumefaciens to deliver synthesized genes to plant cells and induce their expression. It was determined that co-expression of the molecular folding chaperone calreticulin is necessary for LUJV GP-C∆TM to accumulate at detectable levels. Co-expression of an oligossacharyl transferase LmSTT3D, isolated from Leishmania major, may increase the glycan occupancy of the LUJV GP-C∆TM protein, indicated by a molecular mass shift. However, further experimental lines of evidence are needed -such as glycosylation mapping to determine if this is the case. Other parameters such as the optimal day of protein harvest and optical density of recombinant Agrobacterium tumefaciens strains were recorded. Collectively these findings serve as a prototype pipeline for the production of commercially relevant immunogens, diagnostic tools and virus-like particles. This study narrows the focus for bottlenecks in plant protein production, not just for Lujo virus proteins, but for arenaviruses generally and potentially also other haemorrhagic fever-causing viruses. Future efforts should be directed towards addressing the barriers to plant production of complex viral antigens, and to further investigate the utility of mammalian cell-produced LUJV GPC∆TM in animal studies. 2023-07-04T11:07:25Z 2023-07-04T11:07:25Z 2023 2023-07-04T11:06:44Z Master Thesis Masters MSc http://hdl.handle.net/11427/38017 eng application/pdf Department of Molecular and Cell Biology Faculty of Science |
| spellingShingle | Molecular and Cell Biology Isaacs, Abdul Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| thesis_degree_str | Master's |
| title | Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| title_full | Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| title_fullStr | Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| title_full_unstemmed | Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| title_short | Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools |
| title_sort | engineering the nicotiana benthamiana secretory pathway for the production of lujo virus vaccines and diagnostic tools |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/38017 |
| work_keys_str_mv | AT isaacsabdul engineeringthenicotianabenthamianasecretorypathwayfortheproductionoflujovirusvaccinesanddiagnostictools |