Browsing by Author "Meyers, Ann Elizabeth"
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- ItemOpen AccessExpression and characterization of plant produced AHSV nanoparticles encapsulating EGFP(2024) Martins, Angelo; Meyers, Ann Elizabeth; Rybicki EdwardVirus-like particles (VLPs) are virus-based nanoparticles that resemble the native virion but do not contain the viral genome. Heterologous expression of the viral structural proteins results in the spontaneous self-assembly of VLPs that have an empty inner cavity. These particles have an organized, repetitive structure that is very amenable to modification. One can take advantage of this property and fuse a foreign molecule to one of the viral structural proteins and upon VLP self-assembly this foreign molecule may be encapsulated within the empty inner cavity of the VLP. One can also take advantage of the high tailorability of VLPs and functionalize the external surface with targeting ligands for delivery purposes. Alternatively, the structural proteins that form VLPs can have inherent amino acid motifs with a natural targeting affinity for receptors that may be overexpressed on the surface of certain types of cells, for example cancer cells. These properties impart VLPs with great potential as delivery vehicles of diagnostic and/or therapeutic molecules. Core-like particle (CLPs) are a derivative of VLPs that share all the same properties and modification potential, however unlike VLPs, CLPs are comprised of only the viral structural proteins that form the inner capsid layer or ‘core' of the native virion. Herein, two cloning techniques have been used to successfully fuse a fluorescent protein, enhanced green fluorescent protein (EGFP), to the VP3 structural protein of African horse sickness virus (AHSV). Plant-based transient expression of the AHSV EGFP-VP3 and AHSV VP7 structural proteins has been used to generate CLPs from N. benthamiana leaves that successfully encapsulate the EGFP protein. However, interaction of this particle with the human integrin receptor, αvβ3, a receptor that is commonly found overexpressed on the surface of cancer cells could not be confirmed. These results highlight the potential of AHSV CLPs as a cargo carrier, but more research is required to elucidate its potential as a delivery vehicle
- ItemOpen AccessInvestigation of particulate Bluetongue virus vaccines made in plants(2023) Gwynn, Abigail; Meyers, Ann ElizabethBluetongue 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.
- ItemOpen AccessThe Development of an African Horse Sickness Virus VP7 Quasi-Crystal Vaccine Candidate in N. benthamiana(2019) Fearon, Shelley Helen; Meyers, Ann Elizabeth; Hitzeroth, Inga; Rybicki, EdAfrican 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.
- ItemOpen AccessThe Expression of Chikungunya Virus Envelope 2 Glycoprotein Variants in Nicotiana benthamiana for the Development of a Diagnostic Reagent(2020) Naude, Jason Christopher Delville; Meyers, Ann Elizabeth; Chabeda, AleyoChikungunya fever is a non-fatal but highly debilitating disease that affects primates, birds and humans. The causative agent is the chikungunya virus (CHIKV), an arbovirus of the Alphavirus genus. CHIKV is responsible for the largest epidemic recorded for an Alphavirus, infecting an estimated 1.4 to 6 million patients worldwide. Furthermore, it has been recognised by the United States army as a potential biological weapon used for bioterrorism owing to the potential for infection via aerosol. CHIKV is primarily transmitted by infected Aedes aegypti mosquitos and is currently distributed in Africa, parts of Asia and South, Central and North America. As a result of the virus genetically adapting to infect the Aedes albopictus mosquito, its recent and rapid spread to non-endemic regions has occasioned increasing anxiety as well. Infection in humans presents as a sudden onset of fever, rash and severe arthralgia that persists for years. At present, there is no fast and effective diagnostic test to distinguish CHIKV from other similar viruses. This is a problem because viral infection displays the same symptoms as that of dengue, Zika, Ebola and yellow fevers while prognosis, patient care, and persistent symptoms of these viruses are very different. Usually, during the development of a diagnostic reagent, Biosafety Level 3 (BSL3) containment is required for purifying antigens from live viruses. These lab diagnostic tests are expensive to perform and, in regions facing a CHIKV epidemic, are inefficient due to their long waiting periods. This results in patients going undiagnosed or misdiagnosed and/or falling outside the window of prophylactic treatment. As such, a cheap and rapid diagnostic reagent to detect the presence of CHIKV antibodies would be most advantageous. In this study, two recombinant variants of the CHIKV E2 glycoprotein were expressed in Nicotiana benthamiana plants to assess their viability for use in a diagnostic reagent for CHIKV infection. Two versions of a tobacco sp. codon-optimised, 6xhis-tagged CHIKV E2 envelope glycoprotein gene were synthesised and cloned into the plant expression vector, pTRAkc-ERH. The E2 glycoprotein is a desirable protein candidate used for a diagnostic reagent as it is a major target for neutralizing antibody production against CHIKV during early infection. One variant contained a ~52 kDa full length E2 glycoprotein (CHIKV E2-HIS) while the other contained a ~49 kDa truncated E2 glycoprotein lacking its transmembrane domain (CHIKV E2ΔTM-HIS). Following this, an expression time trial was performed whereby the recombinant proteins were expressed in N. benthamiana plants via Agrobacterium-mediated small-scale 6 syringe-infiltration at different optical densities, OD600 = 1.0 and 0.5. To improve expression, both genes were co-infiltrated and co-expressed with a human chaperone proteins calreticulin (CRT) or calnexin (CNX), or a plant silencing-suppressor protein NSs. Expression of the recombinant protein variants alone showed low to undetectable levels of expression in plant leaves across 7 days post infiltration (dpi) for both ODs tested. CHIKV E2ΔTM-HIS yielded the highest levels of all combinations tested at an OD600 = 1.0 when co-expressed with CRT and harvested at 3 dpi. These parameters were used for subsequent scaling up and production of E2 using vacuum infiltration. Attempts at purifying CHIKV E2ΔTM-HIS proteins using Ni-NTA affinity chromatography and further investigation into the exposure of the 6xHis-tag on the native conformation of CHIKV E2ΔTM-HIS indicated that the 6xHis-tag was insufficiently exposed on E2 and thus inaccessible to facilitate purification by Ni-NTA affinity chromatography. Further attempts at purifying recombinant CHIKV E2ΔTM-HIS proteins by pH purification were also unsuccessful as large amounts of plant-protein contaminants present in all samples prevented adequate separation from CHIKV E2ΔTM-HIS. A different approach utilising ammonium sulphate precipitation facilitated separation of recombinant CHIKV E2ΔTM-HIS from some of the contaminating plant proteins in the 30 - 60% ammonium sulphate fraction; however, large amounts of recombinant CRT were co-purified with E2 in this fraction. Although expression of a candidate diagnostic reagent in plants for detecting CHIKV antibodies in the form of E2 glycoprotein was achieved, further research needs to be done to optimise a purification strategy for CHIKV E2ΔTM-HIS proteins.
- ItemOpen AccessThe isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana(2020) Ndlovu, Siphumelele; Meyers, Ann Elizabeth; Meggersee Rosemary; Rybicki, EdMonoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.
- ItemOpen AccessThe production of foot-and-mouth disease virus-like particles in the plant Nicotiana benthamiana: a potential candidate vaccine for foot-and-mouth disease(2018) O'Connor, Steven Patrick; Meyers, Ann Elizabeth; Rybicki, EdwardFoot and mouth disease virus (FMDV) infects cloven-hoofed animals causing the highly contagious foot and mouth disease. It is spread by contact or through aerosol. The disease is often debilitating for infected animals and can be fatal. Severe measures are taken to contain outbreaks; quarantine and trade restrictions are imposed and herds with infected individuals are culled to prevent the spread of the disease. Consequently, outbreaks of the disease have drastic implications for agriculture and social economies which can be devastating for affected countries. There are seven serotypes of the virus; of which SAT1, SAT2, and SAT3 are endemic to Africa. South African buffalo populations such as those in the Kruger National Park, are natural carriers of FMDV (Thomson 1995). Careful monitoring and regular vaccination are necessary to detect and prevent outbreaks and the spread of the disease to livestock of neighbouring areas and farms. The vaccines currently used are inactivated FMDV virions. These are produced in cell culture, an expensive process that requires high levels of biosafety. Furthermore, inactivated virions present non-structural proteins (NSPs) and thus cannot be distinguished from the infectious virus by imported ELISA kits that utilise the NSPs as coating antigens and conventionally produced detecting antibodies. We aimed to use recombinant constructs encoding the FMDV capsid and protease genes, cloned into the different vectors; pRIC, pEAQ and pTRAc, for transient expression in Nicotiana benthamiana to generate virus-like particles as an alternative vaccine candidate. Using a plant based expression system presents numerous advantages over the traditional cell culture production of the vaccine currently used. After having synthesised the FMDV genes P12A and 3C, the fusion gene P1-2A-3C (required for the vaccine) was cloned into these different plant expression vectors available in our laboratory. With Agrobacteria mediated infiltration of N. benthamiana, we demonstrated expression of recombinant protein by western blotting; and Coomassie stain, for each of the different constructs. Analytical ultra-centrifugation through a sucrose gradient was used to purify protein extracts. Comparison against a dilution series of bovine serum albumin was used to quantify the yield for each respective vector construct by densitometry. Transmission Electron Microscopy (TEM) imaging was used to qualitatively determine virus-like particle (VLP) assembly. In conclusion, we demonstrate proof of concept for a viable alternative approach for the production of a candidate vaccine for FMDV.