title: Applications of polyhydroxy butyrate bead technology
Creators: name: Evert, Benjamin J
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Bioengineering
subject: endotoxin-free production
subject: biopolymer particles
subject: protective immunity
subject: bioseparation resin
subject: protein purification
subject: PHA synthase
subject: spytag
subject: spycatcher

description: Recent research into bioengineered polyhydroxy butyrate (PHB) bead technology has resulted in the ability to generate functional polyester nanoparticles and materials with diverse applications across many fields of science. Through protein engineering and by hijacking Escherichia coli as a high yielding cell factory the Rehm lab group has been able to generate hundreds of PHB beads coated in various target proteins for multiple applications. The possible applications of this technology are only limited by our own ideas for targets which can be applied to the PHB bead technology. PHB beads coated in antigenic proteins have been previously developed and used as particulate vaccines in various animal trials. Several other PHB beads have been developed which had demonstrated applications in enzyme immobilisation, bioseperation, diagnostic imaging, drug delivery and bioremediation. The application of PHB bead technology depends on the target proteins designed to coat the surface of the beads. Here I continue exploring novel applications of PHB bead technology bygenerating several novel PHB beads with applications as particulate vaccines and a bioseperation resin which are outlined in this thesis. There are four separate projects highlighted in this thesis, all focused on PHB beads, but each with a different focus and overall aim. The first project I generated several malaria vaccine candidates (Chapter 3 and 4) with the overall aim to generate promising malaria vaccine candidates and highlight the benefits of PHB bead technology as well as explore alternative methods for coating PHB beads with antigenic proteins. Two of the vaccines generated strong immune responses in sheep animal trials and antibodies which were able to inhibit traversal of malaria sporozoites into human hepatocytes. Another vaccine showed promising results in a rat animal trial and will be explored further by future students. The results from this project resulted in one first author manuscript currently submitted for publication and future work to be continued by other students. The second project I generated a SARS-CoV-2 vaccine candidate by using an alternative method of coating PHB beads with the target antigen (Chapter 5). The aim was to fill the unmet need for a rapidly adaptable, scalable, and economically viable vaccine platform technology which could combat the ongoing pandemic and additionally highlight the benefits of a new method for attaching antigenic proteins to PHB beads. This vaccine candidate was combined with six others in two animal trails All six vaccines showed a strong and specific immune response in the first animal study. The two best performing vaccines were assessed by a second animal trial and demonstrated protective immunity in hamsters. The results from this project resulted in a second author manuscript currently submitted for publication.The third project I designed three chikungunya vaccine candidates (Chapter 6). We aimed to generate a chikungunya vaccine candidate that generated a strong and functional immune response and again highlight the benefits of a new method for attaching target proteins to PHB beads. However due to experimental complications and time constraints I was only able to generate one of the three vaccine candidates. This vaccine candidate was characterised and assessed for suitability to be taken into animal trials. The work from this project will be continued by future students. The vaccine candidates will eventually be tested in animal trials. The fourth project I generated three novel bioseperation resins using PHB bead technology (Chapter 7). We aimed to generate a bioseperation resin that was easy to use, improved on current technologies and could be a valuable tool used by scientist in the molecular biology field. The physical and chemical properties of the resin was characterised. The ability to purify three structurally and functionally diverse target proteins was assessed, and the performance was compared to the commonly used His-tag affinity resin. We found that the resin was able to purify the target proteins from complex mixtures even at concentrations not detectable by SDS-PAGE analysis. Furthermore, the resins performance was comparable to the traditionally used His-Tag affinity resin with several distinct advantages. The results from this work resulted in a first author publication. Overall, the work presented in this thesis significantly contributes to the field by furthering the applications and possible applications of bioengineered PHB bead technology. Each of these chapters demonstrates a unique application of PHB bead technology that had previously not been explored. Furthermore, by providing an alternate way of attaching target proteins to PHB beads we open the door to future projects that previously could not be done due to the limitations of the current technology.