The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana
| dc.contributor.advisor | Meyers, Ann Elizabeth | |
| dc.contributor.advisor | Hitzeroth, Inga | |
| dc.contributor.advisor | Rybicki, Ed | |
| dc.contributor.author | Fearon, Shelley Helen | |
| dc.date.accessioned | 2020-04-23T06:53:13Z | |
| dc.date.available | 2020-04-23T06:53:13Z | |
| dc.date.issued | 2019 | |
| dc.date.updated | 2020-04-23T01:17:07Z | |
| dc.description.abstract | African horse sickness (AHS) is a debilitating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS and the commercially used live-attenuated vaccines (LAVs) have various side effects. Insoluble particulates have been shown to increase immunogenicity when compared to soluble subunit vaccines and previous studies demonstrated protection of BALB/c mice immunised with African horse sickness virus (AHSV) VP7 against a lethal challenge of AHSV-7 (Bailey 2016; Rutkowska et al. 2011; St Clair et al. 1999; Storni et al. 2005; Wade-Evans et al. 1997). This study investigates a safer monovalent vaccine alternative based on plant-produced quasicrystals of the serogroup-specific AHSV structural protein, VP7. AHSV serotype 5 (AHSV-5) VP7 was expressed in Nicotiana benthamiana by means of Agrobacterium-mediated infiltration of plant expression vector pRIC3.0 encoding VP7 and quasi-crystals purified by means of density gradient ultracentrifugation. The presence of AHSV VP7 quasi-crystals was confirmed by western immunoblotting with anti-AHSV VLP guinea-pig serum and characterized using transmission electron microscopy. After optimizing the purification protocol and achieving satisfactory concentrations, AHSV-5 VP7 quasi-crystals were used in guinea-pig immunogenicity studies where the experimental group (n=5) was inoculated with prime- and boostinoculations of between 10 and 50 µg of purified AHSV VP7 quasi-crystals, and the control group (n=5) inoculated with a control inoculum prepared in the identical manner as the vaccine but using a pRIC3.0 expression vector lacking VP7. Western immunoblot analysis of the humoral response showed stimulation of very high titres of anti-VP7 antibodies 28 days after the boost-inoculation. In addition, RNA-seq transcriptome profiling of guinea-pig spleen derived RNA was used to investigate the global immune response to AHSV-5 VP7 quasi-crystals. Thirty genes involved in innate and adaptive immunity were found to be significantly differentially expressed (q≤0.05) in experimental transcriptome data when compared to the control. Differential expression of genes involved in T-helper (Th)1, Th2 and Th17 cell differentiation and the T-cell receptor signalling pathway suggest a possible cell-mediated immune response to AHSV-5 VP7 quasi-crystals. Upregulation of several important cytokines and cytokine receptors were noted in response to VP7 quasi-crystals e.g. TNFSF14, CX3CR1, IFNLR1 and IL17RA. TNFSF14 and CX3CR1 play a role in T-cell proliferation and cytotoxic T-cell responses respectively. And IFNLR1 and IL17RA are key cytokines in antiviral defences. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. To the best of our knowledge, this study is the first to report the expression of plantproduced AHSV VP7 quasi-crystals and the first time that the cell-mediated immune response to these particles has been assessed. While further investigation is needed, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic, inducing both humoral and cell-mediated responses. | |
| dc.identifier.apacitation | Fearon, S. H. (2019). <i>The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana</i>. (). ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from | en_ZA |
| dc.identifier.chicagocitation | Fearon, Shelley Helen. <i>"The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana."</i> ., ,Faculty of Science ,Department of Molecular and Cell Biology, 2019. | en_ZA |
| dc.identifier.citation | Fearon, S.H. 2019. The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana. . ,Faculty of Science ,Department of Molecular and Cell Biology. | en_ZA |
| dc.identifier.ris | TY - Thesis / Dissertation AU - Fearon, Shelley Helen AB - African horse sickness (AHS) is a debilitating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS and the commercially used live-attenuated vaccines (LAVs) have various side effects. Insoluble particulates have been shown to increase immunogenicity when compared to soluble subunit vaccines and previous studies demonstrated protection of BALB/c mice immunised with African horse sickness virus (AHSV) VP7 against a lethal challenge of AHSV-7 (Bailey 2016; Rutkowska et al. 2011; St Clair et al. 1999; Storni et al. 2005; Wade-Evans et al. 1997). This study investigates a safer monovalent vaccine alternative based on plant-produced quasicrystals of the serogroup-specific AHSV structural protein, VP7. AHSV serotype 5 (AHSV-5) VP7 was expressed in Nicotiana benthamiana by means of Agrobacterium-mediated infiltration of plant expression vector pRIC3.0 encoding VP7 and quasi-crystals purified by means of density gradient ultracentrifugation. The presence of AHSV VP7 quasi-crystals was confirmed by western immunoblotting with anti-AHSV VLP guinea-pig serum and characterized using transmission electron microscopy. After optimizing the purification protocol and achieving satisfactory concentrations, AHSV-5 VP7 quasi-crystals were used in guinea-pig immunogenicity studies where the experimental group (n=5) was inoculated with prime- and boostinoculations of between 10 and 50 µg of purified AHSV VP7 quasi-crystals, and the control group (n=5) inoculated with a control inoculum prepared in the identical manner as the vaccine but using a pRIC3.0 expression vector lacking VP7. Western immunoblot analysis of the humoral response showed stimulation of very high titres of anti-VP7 antibodies 28 days after the boost-inoculation. In addition, RNA-seq transcriptome profiling of guinea-pig spleen derived RNA was used to investigate the global immune response to AHSV-5 VP7 quasi-crystals. Thirty genes involved in innate and adaptive immunity were found to be significantly differentially expressed (q≤0.05) in experimental transcriptome data when compared to the control. Differential expression of genes involved in T-helper (Th)1, Th2 and Th17 cell differentiation and the T-cell receptor signalling pathway suggest a possible cell-mediated immune response to AHSV-5 VP7 quasi-crystals. Upregulation of several important cytokines and cytokine receptors were noted in response to VP7 quasi-crystals e.g. TNFSF14, CX3CR1, IFNLR1 and IL17RA. TNFSF14 and CX3CR1 play a role in T-cell proliferation and cytotoxic T-cell responses respectively. And IFNLR1 and IL17RA are key cytokines in antiviral defences. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. To the best of our knowledge, this study is the first to report the expression of plantproduced AHSV VP7 quasi-crystals and the first time that the cell-mediated immune response to these particles has been assessed. While further investigation is needed, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic, inducing both humoral and cell-mediated responses. DA - 2019 DB - OpenUCT DP - University of Cape Town KW - Molecular and Cell Biology LK - https://open.uct.ac.za PY - 2019 T1 - The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana TI - The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana UR - ER - | en_ZA |
| dc.identifier.uri | https://hdl.handle.net/11427/31671 | |
| dc.identifier.vancouvercitation | Fearon SH. The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana. []. ,Faculty of Science ,Department of Molecular and Cell Biology, 2019 [cited yyyy month dd]. Available from: | en_ZA |
| dc.language.rfc3066 | eng | |
| dc.publisher.department | Department of Molecular and Cell Biology | |
| dc.publisher.faculty | Faculty of Science | |
| dc.subject | Molecular and Cell Biology | |
| dc.title | The Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana | |
| dc.type | Master Thesis | |
| dc.type.qualificationlevel | Masters | |
| dc.type.qualificationname | MSc |