Investigation of particulate Bluetongue virus vaccines made in plants

Gwynn, Abigail

Investigation of particulate Bluetongue virus vaccines made in plants

Thesis / Dissertation

2023

Abstract

Bluetongue virus (BTV) infects ruminants but predominantly causes severe and often fatal haemorrhagic fever, known as Bluetongue (BT) disease, in sheep. Increasing global temperatures have contributed to the global dissemination of BTV. This is a result of the Culicoides insect vectors which function optimally in warm and wet climates. In South Africa, the eradication and control of BTV is made difficult due to the circulation of 21 of the 28 known serotypes and their limited serological cross-reactivity. Current vaccine strategies include live attenuated and inactivated vaccines. Although they have been successful in protecting animals, there are many limitations and risks associated with these vaccine strategies such as reversion to virulence, re-assortment and short-lasting immunity. There is thus a need for vaccines that are safe, scalable, economically viable and effective against multiple serotypes. A variety of recombinant BTV vaccine strategies have been developed which address and improve upon some of the limitations of the commercially available vaccine strategies. One of the most promising strategies is that of the virus-like particle (VLP). BTV VLPs comprise four structural proteins, namely VP2, VP3, VP5 and VP7. VP2 is highly immunogenic and is responsible for eliciting neutralising antibodies against the virus. Although a number of different BTV VLPs have been produced in traditional protein expression systems, such as insect cells, these production methods are considered too expensive to compete with those of the commercial vaccines. This has led to the consideration of plants as an alternative vaccine expression system. Plants are easily scaled up, upstream processes are cost effective, they are easy to work with, and do not require sterile environments and expensive infrastructure to maintain. A number of studies have shown that when the four major BTV structural proteins are transiently co-expressed in Nicotiana benthamiana, they self-assemble into VLPs which can be used as vaccines to protect animals against homologous serotypes. The aim of this study was to develop and compare two different particulate BTV candidate vaccines made in plants and determine their ability to elicit specific immunity in guinea pigs. The first vaccine approach was to develop a chimeric BTV VLP vaccine. This was achieved by substituting the immunogenic tip domain of the VP2 gene of BTV serotype 8 (BTV8) with that of the corresponding domain of BTV serotype 1 (BTV1) generating a chimeric VP2 which, when co-expressed in plants with the remaining BTV8 VP3, VP5 and VP7, resulted in chimeric BTV1/8 VLPs. The second approach involved the display of the immunogenic BTV1 VP2 tip domain on a capsid protein particle. Here, the domain was displayed on the surface of the bacteriophage AP205 particle through the application of the SpyTag (ST)/SpyCatcher (SC) bioconjugation method. It was anticipated that these vaccine candidates would be safe to use and allow for rapid production and scalability. Moreover, only a small fragment of the BTV8 VP2 gene would need to be modified to allow for a VLP to be made against a new serotype. Chimeric BTV1/8 VLP protein expression in plants, VLP extraction, and purification protocols were optimised using previously made homologous BTV8 VLPs for comparison. Plants were vacuum coinfiltrated with the chimeric BTV1/8 VP2 protein and the BTV8 VP3, VP5 and VP7 proteins. Protein was extracted from the plants and VLPs were subsequently purified by density gradient ultracentrifugation. The VLP proteins were detected on Western blots and Coomassie-stained gels. These methods were optimised to maximise protein yields by increasing the salt concentration of the extraction and purification buffer, maintaining an alkaline pH throughout the extraction and maturation process and harvesting at five days post-infiltration. Transmission electron microscopy (TEM) confirmed the presence of a mixture of core-like particles (CLPs), assembly intermediates and fully formed VLPs. These optimised methods were sufficient to produce high enough yields of BTV8 and BTV1/8 VLPs with protein yields of 35mg/kg fresh leaf weight (FLW) and 34mg/kg FLW, respectively, to be used in immunogenicity trials in guinea pigs. The alternative vaccine strategy involved the display of the BTV1 VP2 tip domain on phage AP205 particles. We utilised the ST/SC antigen display technology for the display of the BTV1 antigenic tip domain on the surface of the AP205 capsid. ST was fused to the N-terminal of the AP205 protein (STAP205) while SC was fused to the C-terminal of the BTV1 VP2 tip domain used in the chimeric VLPs (BTV1Tip-SC). Both components were expressed in plants and extracted and purified separately before combining for in vitro coupling. The ST-AP205 particles were purified by density gradient ultracentrifugation while the BTV1Tip-SC proteins were purified by nickel affinity chromatography. The purified components were coupled in vitro in a molar ratio of 1:3 (ST-AP205:BTV1Tip-SC). The 60kDa coupled complex was detected on Western blots and Coomassie gels with an estimated protein concentration of approximately 0.03ug/uL and a coupling efficiency of 44%. Finally, since there was insufficient coupled protein product for immunisation doses, only the immunogenicity of the plant produced chimeric BTV1/8 VLPs compared with the BTV8 VLPs were tested. Five guinea pigs per vaccine group (BTV8 VLPs and chimeric BTV1/8 VLPs) were immunised with 15ug of the appropriate vaccine and boosted 13 days later. Serum was collected 41 days post immunisation and used to determine whether there was an immunogenic response to the vaccines by Western blotting and indirect enzyme-linked immunosorbent assays (ELISAs). This preliminary immunogenicity trial found that both VLP candidate vaccines induced an immune response in guinea pigs. While the BTV1 VP2 tip display vaccine strategy still requires further optimisation to generate more dose-appropriate yields, the VLP vaccine strategy tested here shows great potential for further development into a BTV vaccine candidate that is safe, scalable and has potential for multivalency.

Keywords

Molecular and Cell Biology

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