Browsing by Author "Dennis, Susan Jennifer"

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    Development of plant-produced African horse sickness vaccines
    (2019) Dennis, Susan Jennifer; Rybicki, Ed; Hitzeroth, Inga; Meyers, Ann
    African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades, but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines, initially focusing on the use of insect and mammalian cell expression systems. More recently, several studies have demonstrated the potential for using plant expression systems for the production of virus-like particles (VLPs), which are excellent vaccine candidates, as they do not contain virus genetic material and are DIVA compliant. A vaccine alternative to the currently used live vaccine necessarily needs to provide protection against all nine serotypes of the virus. Cross-protection has been shown to exist between certain serotypes of the virus and as capsid protein VP2 is the protein responsible for AHSV serotype specificity, the idea of a plant-produced VLP vaccine containing a representative VP2 protein from each of the different serotype groups, was conceived. Such a vaccine would potentially provideprotection against all 9 serotypes of the virus and would have DIVA capability. Furthermore, it would address local concerns regarding the use of a live vaccine and would serve as a potentially acceptable prophylactic or rapid response antidote in the wider international context. This work describes two approaches in the development of VLP vaccines in plants. In the first part of this study, the ability of 2 different serotypes of plant-produced AHSV VLPs to safely stimulate an immune response in horses, was investigated. Co-infiltration of Nicotiana benthamiana plants with Agrobacterium constructs encoding the four AHSV serotype 5 structural proteins VP2, VP3, VP5 and VP7, was shown to result in assembly of complete VLPs. Furthermore, co-infiltration with the constructs, encoding VP3 and VP7, together with constructs encoding the two outer capsid proteins VP2 and VP5 of a second serotype, AHSV 4, resulted in assembly of complete AHSV 4 VLPs. Horses vaccinated with plant-produced AHSV 4 and 5 VLPs, all seroconverted after two doses of the vaccine and the virus neutralization titres indicated that the plant-produced VLP vaccines are likely to be at least as effective as the current LAV in protecting against AHSV 4 or AHSV 5. However, they have the added advantage of being free from any of the associated risks of a live vaccine, such as reversion to virulence or genetic re-assortment with field or vaccine strains. In the second part of the study, the use of the so-called SpyTag/SpyCatcher or bacterial “superglue” technology was investigated. This technology is based on the peptide SpyTag irreversibly coupling to the SpyCatcher protein, forming an isopeptide bond when the two are mixed together. The plant-based expression system was used to produce Spy VLPs consisting of either Acinetobacter phage (AP205) VLPs or tobacco mosaic virus (TMV) VLPs displaying a SpyTag or SpyCatcher peptide. In addition, AHSV 5 VP2 displaying SpyTag was expressed in plants and several coupling strategies were tested to determine whether AP205 particles displaying AHSV 5 VP2 could be formed as a result of binding between the SpyTag/SpyCatcher moieties of the recombinant proteins. Although it was not proven that coupling occurred, this research will pave the way towards developing a multivalent vaccine platform whereby VP2 of different AHSV serotypes can be displayed on the Spy VLP surface to allow optimal presentation of these proteins to the animal's immune system. Together, the results obtained in this study show that there is great potential for the production of novel, diverse, efficacious and economically viable AHSV VLP vaccines in plants.
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    Investigating the production of a particulate plant-produced vaccine candidate against African horse sickness
    (2021) Luhanga, Gloria; Hitzeroth, Inga; Meyers, Ann; Dennis, Susan Jennifer
    African horse sickness (AHS) is a non-communicable, infectious disease that affects equids and is mainly prevalent in sub-Saharan Africa. The disease has a major impact on the economy of the equine industry as well as an emotional impact on horse owners. There are nine known serotypes of African horse sickness virus (AHSV), which is spread by Culicoides midges. Currently, a multivalent live attenuated vaccine is the only vaccine licensed for use in South Africa. However, it has the inherent risk of reverting to virulence as well as the possibility of genome segment reassortment between vaccine and outbreak strains. Additionally, it is not DIVA compliant (cannot Differentiate between Infected and Vaccinated Animals). There is therefore a need for a safer and more cost-effective alternative vaccine to protect horses against AHSV. Virus-like particles (VLPs) that display antigens on their surface may be suitable vaccine platforms. One such display particle is the phage AP205 VLP, which is comprised of AP205 coat proteins. To aid antigen display, studies have utilized the SpyTag (ST) - SpyCatcher (SC) or “plug-and-display” system, a novel conjugation system used to display several antigens fused to AP205 VLPs. This study aimed at displaying the neutralizing epitope of AHSV serotype 5, known as the VP2 domain (dom) (873bp), on phage AP205 VLP particles using the SpyTag/SpyCatcher technology. The display particle vaccine candidates were produced in Nicotiana benthamiana plants. Firstly, AHSV 5 VP2dom was expressed by being linked to either the ST or SC peptide at its C-terminus. Recombinant pEAQ-AHSV 5-VP2dom ST and pEAQ-AHSV 5- VP2domSC plasmid constructs were constructed from the full-length pEAQ-AHSV 5-VP2- SpyTag and pEAQ-AHSV 5-VP2-SpyCatcher clones using in-fusion cloning. The ST/SC constructs were transformed into Stellar™ competent E. coli cells and thereafter into Agrobacterium tumefaciens AGL-1 cells. Expression time trials were conducted on plants infiltrated with the recombinant Agrobacterial strains to examine transient AHSV 5- VP2domST/SC small-scale expression. Expression was detected for AHSV 5 VP2domSC but not AHSV 5 VP2domST using guinea pig anti-AHSV 5 and rabbit antiST-AP205 sera. Secondly, the development of a particle display vaccine candidate was investigated by coupling AHSV 5 VP2domSC to plant-expressed ST-AP205 VLPs. Three coupling techniques, namely in vitro coupling of purified products, co-infiltration and co-purification, were deployed to determine the assembly of ST-AP205_AHSV 5 VP2domSC VLPs. In vitro coupling involved carrying out separate infiltrations and purification of pEAQ-STAP205 VLPs and pEAQ-AHSV 5 VP2domSC in plants and thereafter mixing the purified products. For co-infiltration, pEAQ-ST-AP205 VLPs and VP2domSC recombinant cultures were used together to infiltrate plants and the presence of complex formations was determined. During co-purification, the presence of coupled products was analysed following separate infiltration of plants with pEAQ-ST-AP205 and VP2domSC recombinant Agrobacterial strains; the homogenates were then incubated together at different VLP: antigen leaf:weight ratios. From the three coupling techniques, copurification at a 1:1 VLP: antigen ratio was identified as the best coupling approach based on the quality and quantity of particles visualised by electron microscopy. These findings indicate the potential of producing an AHSV vaccine candidate in plants, which is ultimately a safer and cheaper alternative to the currently-produced AHSV vaccine. Moreover, this preliminary data may pave the way for developing a vaccine that provides protection against all nine serotypes of AHSV by displaying VP2domSC for other serotypes on the ST-AP205 display particles.