Browsing by Author "Pietersen, Inge"
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- ItemOpen AccessNovel Production of Bovine Papillomavirus Pseudovirions in Tobacco Plants(2020-11-28) Pietersen, Inge; van Zyl, Albertha; Rybicki, Edward; Hitzeroth, IngaVaccine efficacy requires the production of neutralising antibodies which offer protection against the native virus. The current gold standard for determining the presence of neutralising antibodies is the pseudovirion-based neutralisation assay (PBNA). PBNAs utilise pseudovirions (PsVs), structures which mimic native virus capsids, but contain non-viral nucleic material. PsVs are currently produced in expensive cell culture systems, which limits their production, yet plant expression systems may offer cheaper, safer alternatives. Our aim was to determine whether plants could be used for the production of functional PsVs of bovine papillomavirus 1 (BPV1), an important causative agent of economically damaging bovine papillomas in cattle and equine sarcoids in horses and wild equids. BPV1 capsid proteins, L1 and L2, and a self-replicating reporter plasmid were transiently expressed in Nicotiana benthamiana to produce virus-like particles (VLPs) and PsVs. Strategies to enhance particle yields were investigated and optimised protocols were established. The PsVs’ ability to infect mammalian cells and express their encapsidated reporter genes in vitro was confirmed, and their functionality as reagents in PBNAs was demonstrated through their neutralisation by several different antibodies. This is the first report of BPV PsVs expressed in plants and demonstrates the potential for the development of therapeutic veterinary vaccines in planta.
- ItemOpen AccessWorking towards a plant-produced Bovine papillomavirus vaccine: expression of BPV1 virus-like particles and pseudovirions in Nicotiana benthamiana(2019) Pietersen, Inge; Hitzeroth, Inga; van Zyl, Alta; Rybicki, EdBovine papillomaviruses (BPVs) are DNA viruses implicated in several diseases and cancers of considerable veterinary and agricultural importance in cattle, horses, as well as several wild animal species, leading to their economic depreciation through the deterioration of their function and appearance. As BPV is widespread, easily transmissible, and infection often occurs asymptomatically, it is imperative that interventions are carried out to mitigate the threat of outbreaks occurring in livestock. There is a demand for prophylactic vaccines to immunise animals routinely and from a young age so as to prevent infection from occurring, as there is currently no fully effective treatment against established disease. Virus-like particles (VLPs) have in recent years been adopted as safe and efficacious vaccine antigens, and pseudovirions (PsVs) as experimental vaccines and vectors for therapeutic agents. Prophylactic VLP-based candidate vaccines against BPV types 1, 2, and 4 have previously been produced, yet these produce antibodies which are largely type-specific, and are expensive and not widely available in developing countries, where food security and the use of cattle for meat, dairy, leather products, as well as draft animals, is often essential for survival. Plant-based expression systems for the production of biopharmaceuticals, such as vaccines, offer several advantages over traditional protein expression systems, chief of which is a reduction in production costs, improved biosafety features, and a potential for rapid scalability. BPV1 is one of the most virulent BPV types and, as the most prevalent type, is responsible for the majority of BPV-related diseases. To address the lack of a cheap and readily-available vaccine, BPV1 VLPs and PsVs were expressed in N. benthamiana plants as a prelude to producing a candidate prophylactic and therapeutic vaccine. Expression of L1 and L1/L2 VLPs was successful, as was the encapsidation of a reporter gene plasmid into the L1/L2 VLPs, demonstrating the first successful in planta production of BPV PsVs. Several approaches to optimise VLP and PsV purification and expression were also explored, and significant increases in both protein expression levels and numbers of VLPs and PsVs was observed through the application of each of the following techniques: increasing acetosyringone induction concentrations from 200 μM to 500 μM; a brief heat-shock treatment of plants 2 days post agroinfiltration (dpi); and extending the harvesting time of plant biomass from 4dpi to 6dpi. Furthermore, purification and concentration of BPV1 particles was further enhanced through centrifugation of clarified plant extract on an iodixanol density gradient prepared with a 3M NaCl buffer. PsVs produced through these methods were shown to be capable of pseudo-infecting and expressing the reporter gene in mammalian HEK293TT cells, and neutralisation of the PsVs by different monoclonal antibodies was also demonstrated. These findings highlight the potential use of BPV PsVs for a diverse range of functions, including their potential use as dual prophylactic-therapeutic vaccines, capable of delivering therapeutics or DNA vaccines to specific tissues, as well as their ability to be used as model organisms for the study of HPV and other PVs.