Browsing by Author "Hitzeroth Inga"

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    Development of microalgae as a biopharming platform
    (2019) Els, Johann Hendrik; Rybicki, Edward; Hitzeroth Inga; Harrison, Sue
    Microalgae may be a powerful biopharmaceutical production platform that is still in its infancy of development. The research done in this project tested the feasibility of creating algal cell packs, a novel immobilised microalgae transient production platform for the expression of recombinant protein. First it had to be established whether the available plant expression vectors could be used for the transfer of genetic material into packed microalgae. The method showed successful transfer of the neomycin phosphotransferase II resistance gene (nptII). Further experiments analysed the plant expression vectors pTRAc and pRIC3.0 for expression of enhanced green fluorescent protein (EGFP) in Scenedesmus spp. by western blotting. Possible replication of the plant geminivirus-derived pRIC3.0 was then confirmed by comparing to replication in Nicotiana benthamiana by quantitative polymerase chain reaction (qPCR). Western blot results indicated EGFP expression in N. benthamiana but not in Scenedesmus. By using PCR the presence of EGFP DNA in Scenedesmus was detected but qPCR showed no increase of the pRIC3.0 replicon. Despite no detection via antibodies of EGFP in Scenedesmus, green fluorescence was observed. These initial results showed promise and points to a system that requires optimisation for increased transfection rates and protein expression. Following on from the initial work, the project set out to determine the feasibility of expressing a recombinant anti-Ebola viral inhibitor protein in three different plant based platforms namely N. benthamiana, a microalgal genus, Desmodesmus and a BY2 tobacco plant-cell culture. Protein expression was compared between the Desmodesmus algal cellpack, N. benthamiana plant expression system and BY-2 plant cell packs by western blotting. Four designs of the viral inhibitor fused to the maize ƴ-zein protein body inducing protein, ZERA, were expressed in trace quantities. Transient expression was more prominent in the algal cell packs than in N. benthamiana and BY-2 cells. The algal cell pack system may potentially be a powerful tool to test recombinant protein expression in a range of microalgal hosts via Agrobacterium-mediated genetic transfection. The future development of recombinant protein expression platforms could be enhanced by rapid testing of protein production in different species. Refinement needs to be done on the algal cell pack to increase transfection efficiency and expression in microalgae to produce commercially viable quantities of heterologous protein.
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    Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines
    (2024) Edwards, Amy; van Zyl, Albertha; Hitzeroth Inga
    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.