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Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines
Infection with human papillomavirus (HPV) is the leading cause of cervical cancer, the fourth most common cancer in women globally. Cervical cancer results in an estimated 604,000 new cases and 342,000 deaths each year, with the majority of these cases reported in sub-Saharan Africa. HPV-16 is a hig...
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| Format: | Thesis |
| Language: | Eng |
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Department of Molecular and Cell Biology
2025
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| _version_ | 1867614040379883520 |
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| access_status_str | Open Access |
| author | Edwards, Amy |
| author2 | van Zyl, Albertha |
| author_browse | Edwards, Amy van Zyl, Albertha |
| author_facet | van Zyl, Albertha Edwards, Amy |
| author_sort | Edwards, Amy |
| collection | Thesis |
| description | Infection with human papillomavirus (HPV) is the leading cause of cervical cancer, the fourth most common cancer in women globally. Cervical cancer results in an estimated 604,000 new cases and 342,000 deaths each year, with the majority of these cases reported in sub-Saharan Africa. HPV-16 is a high-risk oncogenic subtype and, along with HPV-18, is associated with >70% of all cervical cancers. While current vaccines can prevent infection with high-risk HPVs, they cannot induce regression of persistent infections, thus the development of vaccines that function therapeutically is required. DNA vaccines are ideal candidates for therapeutic treatment; however, naked DNA vaccines are associated with ineffective presentation to antigen presenting cells (APCs). This limitation can be overcome by using pseudovirions (PsVs) as vaccine delivery vehicles. HPV PsVs are highly immunogenic synthetic viral particles consisting of L1 and L2 capsid proteins, which self-assemble to package pseudogenome DNA. Pre-existing immunity to high-risk HPVs through natural infection and vaccination, however, preclude their use as delivery vehicles for DNA vaccines. The development of non-human papillomaviruses (PVs) PsVs for gene delivery is a novel alternative. PV PsVs are conventionally produced in mammalian cells. However, plants have demonstrated potential as an alternative platform for rapid PsV production due to their scalability and cost-efficiency. Therefore, the aim of this study was to investigate plant-based production of bovine papillomavirus 1 (BPV-1) PsVs encapsidating a HPV-16 therapeutic DNA vaccine candidate, and to assess their infectivity in mammalian cells. Initially, strategies to optimise Agrobacterium-mediated transient expression of BPV-1 L1 and L2 capsid proteins in Nicotiana benthamiana were explored. Approaches such as an increased acetosyringone concentration for recombinant Agrobacterium induction, a heat-shock treatment of post-infiltrated plants, and an extended in planta maturation were investigated. A pseudogenome encoding secreted embryonic alkaline phosphatase (SEAP) was co-infiltrated with BPV-1 L1- and L2-encoding expression vectors. L1 protein expression and particle assembly were confirmed with western blotting and transmission electron microscopy (TEM) respectively. However, no SEAP expression was observed following infection of HEK293TT cells with the plant-made particles and none of the strategies investigated were conclusively found to increase BPV-1 PsV yield. Several geminivirus-derived self-replicating reporter plasmids encoding a HPV-16 shuffled E7 (E7SH) sequence were constructed using In-Fusion cloning. These constructs were co-expressed in N. benthamiana with expression vectors encoding BPV-1 or HPV-35 L1 and L2 proteins. HPV-35 was chosen as it has been shown to encapsidate a Zera®E7SH DNA vaccine in plants for delivery to mammalian cells in vitro. Following purification, rolling circle amplification (RCA) analysis showed successfully encapsidation of the E7SH-based pseudogenomes within the plant-made PsVs. However, TEM showed that PsV yields were low and no E7 expression was observed following infection of HEK293TT cells with the plant-made PsVs. These results indicated that encapsidation efficiency of PsVs produced in plants is low. BPV1 and HPV-35 PsVs were also produced in HEK293TT cells and comparative analyses with plant-made particles revealed that, while the pseudogenomes were successfully encapsidated in the PsVs produced in both systems, only the HEK293TT-made PsVs effectively delivered their packaged DNA into mammalian cells. These findings demonstrate the ability of PsVs to self-assemble and encapsidate pseudogenome DNA in planta while also revealing potential limitations of plant-based PsV production. For plant-made PsVs to reach their potential in gene delivery, further optimisation and characterisation of plant-based PV PsV expression is required. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/40894 |
| institution | University of Cape Town (South Africa) |
| language | Eng |
| last_indexed | 2026-06-10T12:45:43.270Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| 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:11427/40894 Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines Edwards, Amy van Zyl, Albertha Hitzeroth Inga Molecular and Cell Biology Infection with human papillomavirus (HPV) is the leading cause of cervical cancer, the fourth most common cancer in women globally. Cervical cancer results in an estimated 604,000 new cases and 342,000 deaths each year, with the majority of these cases reported in sub-Saharan Africa. HPV-16 is a high-risk oncogenic subtype and, along with HPV-18, is associated with >70% of all cervical cancers. While current vaccines can prevent infection with high-risk HPVs, they cannot induce regression of persistent infections, thus the development of vaccines that function therapeutically is required. DNA vaccines are ideal candidates for therapeutic treatment; however, naked DNA vaccines are associated with ineffective presentation to antigen presenting cells (APCs). This limitation can be overcome by using pseudovirions (PsVs) as vaccine delivery vehicles. HPV PsVs are highly immunogenic synthetic viral particles consisting of L1 and L2 capsid proteins, which self-assemble to package pseudogenome DNA. Pre-existing immunity to high-risk HPVs through natural infection and vaccination, however, preclude their use as delivery vehicles for DNA vaccines. The development of non-human papillomaviruses (PVs) PsVs for gene delivery is a novel alternative. PV PsVs are conventionally produced in mammalian cells. However, plants have demonstrated potential as an alternative platform for rapid PsV production due to their scalability and cost-efficiency. Therefore, the aim of this study was to investigate plant-based production of bovine papillomavirus 1 (BPV-1) PsVs encapsidating a HPV-16 therapeutic DNA vaccine candidate, and to assess their infectivity in mammalian cells. Initially, strategies to optimise Agrobacterium-mediated transient expression of BPV-1 L1 and L2 capsid proteins in Nicotiana benthamiana were explored. Approaches such as an increased acetosyringone concentration for recombinant Agrobacterium induction, a heat-shock treatment of post-infiltrated plants, and an extended in planta maturation were investigated. A pseudogenome encoding secreted embryonic alkaline phosphatase (SEAP) was co-infiltrated with BPV-1 L1- and L2-encoding expression vectors. L1 protein expression and particle assembly were confirmed with western blotting and transmission electron microscopy (TEM) respectively. However, no SEAP expression was observed following infection of HEK293TT cells with the plant-made particles and none of the strategies investigated were conclusively found to increase BPV-1 PsV yield. Several geminivirus-derived self-replicating reporter plasmids encoding a HPV-16 shuffled E7 (E7SH) sequence were constructed using In-Fusion cloning. These constructs were co-expressed in N. benthamiana with expression vectors encoding BPV-1 or HPV-35 L1 and L2 proteins. HPV-35 was chosen as it has been shown to encapsidate a Zera®E7SH DNA vaccine in plants for delivery to mammalian cells in vitro. Following purification, rolling circle amplification (RCA) analysis showed successfully encapsidation of the E7SH-based pseudogenomes within the plant-made PsVs. However, TEM showed that PsV yields were low and no E7 expression was observed following infection of HEK293TT cells with the plant-made PsVs. These results indicated that encapsidation efficiency of PsVs produced in plants is low. BPV1 and HPV-35 PsVs were also produced in HEK293TT cells and comparative analyses with plant-made particles revealed that, while the pseudogenomes were successfully encapsidated in the PsVs produced in both systems, only the HEK293TT-made PsVs effectively delivered their packaged DNA into mammalian cells. These findings demonstrate the ability of PsVs to self-assemble and encapsidate pseudogenome DNA in planta while also revealing potential limitations of plant-based PsV production. For plant-made PsVs to reach their potential in gene delivery, further optimisation and characterisation of plant-based PV PsV expression is required. 2025-02-10T11:06:41Z 2025-02-10T11:06:41Z 2024 2025-02-10T10:59:49Z Thesis / Dissertation Masters MSc http://hdl.handle.net/11427/40894 Eng application/pdf Department of Molecular and Cell Biology Faculty of Science |
| spellingShingle | Molecular and Cell Biology Edwards, Amy Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| thesis_degree_str | Master's |
| title | Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| title_full | Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| title_fullStr | Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| title_full_unstemmed | Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| title_short | Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines |
| title_sort | towards the development of plant made psvs as potential delivery vehicles for therapeutic hpv vaccines |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/40894 |
| work_keys_str_mv | AT edwardsamy towardsthedevelopmentofplantmadepsvsaspotentialdeliveryvehiclesfortherapeutichpvvaccines |