Browsing by Author "Hitzeroth, Inga"

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    Beak and feather disease virus candidate vaccine development
    (2012) Duvenage, Lucian; Rybicki, Ed; Hitzeroth, Inga; Meyers, Ann
    [Fix supervisors field.] Psittacine beak and feather disease, caused by a circovirus known as beak and feather disease virus (BFDV), is a threat to both wild and captive psittacine species. There is currently no vaccine against BFDV and safe and affordable vaccine candidates are needed to alleviate the disease burden caused by this virus. Production of the BFDV's major antigenic determinant, the capsid protein (CP), in the inexpensive and highly scalable plant expression system, could satisfy these requirements as a potential subunit vaccine. In this work, truncated CP (ÄN40 CP) was first expressed in E. coli to successfully generate anti-CP polyclonal antibodies. ÄN40 CP and full-length CP transient expression in tobacco (Nicotiana benthamiana) was optimised as fusions to elastin-like polypeptide (ELP). Fusion of CP or ÄN40 CP to ELPs of different lengths was shown to increase yield relative to unfused CP/ÄN40 CP. Free ELP and a GFP-ELP fusion could be purified by inverse transition cycling (ITC), using centrifugation and membrane filtration methods. A ÄN40 CP-ELP fusion expressed in plants could be partially purified and represents low-cost vaccine candidate against BFDV. A candidate DNA vaccine expressing ÄN40 CP was also evaluated for expression of the antigen in vitro and may prove useful in a prime-boost regimen together with one of the plant-produced vaccine candidates.
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    Development of a plant-made immunoassay for the detection of Porcine circovirus infections in South African swine herds
    (2021) Angobe, Aune Tuyoleni; Hitzeroth, Inga; van Zyl, Albertha
    Porcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirusassociated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication-associated protein. Recombinantly expressed CP can selfassemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLPbased and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study was to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. Production of PCV-2 CP in Nicotiana benthamiana via transient Agrobacterium-mediated expression was optimised by comparing two expression vectors, pEAQ-HT and pCBP2, and VLPs were also expressed in Escherichia coli. VLPs produced in plants and in E. coli were used to set up a lateral flow device. In addition, various purification methods of VLPs such as ion exchange chromatography (IEC) and sucrose gradient ultracentrifugation were explored to obtain pure VLPs free of bacterial contamination. The VLPs were successfully expressed in N. benthamiana with both pEAQ-HT and pCBP2, and VLPs were subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs. To further purify the VLPs IEC was used, and fully assembled VLPs which were free of contamination were prepared. Purified VLPs expressed in plants and E. coli were successfully used as coating antigen in lateral flow devices, which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera. E. coli-made VLPs showed higher affinity to PCV-2 antibodies compared to plant-made VLPs. In conclusion, this study has successfully demonstrated the potential to use a plantbased transient expression system to produce affordable diagnostic reagent, especially for developing countries. This is the first study that expressed PCV-2 VLPs using a pCBP-2 expression vector and used PCV-2 VLPs as a coating reagent in the development of a lateral flow test as a proof of concept.
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    Development of a potential challenge model and plant-produced vaccine candidate for beak and feather disease virus
    (2015) Regnard, Guy Louis; Hitzeroth, Inga; Rybicki, Edward P
    Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). An outbreak of the disease has been reported in wild endangered Cape parrots (Poicephalus robustus), which is endemic to South Africa. No treatment or vaccine is commercially available. In this study, an investigation into the outbreak was undertaken. BFDV diversity was assessed and viral load and clinical signs correlated. A plant-produced BFDV subunit vaccine was produced in parallel with a corresponding challenge model. Cape parrots were assessed and 53 blood samples collected. Viral load was determined using quantitative real-time PCR (qPCR), and 22 BFDV full-length genome sequences acquired to infer phylogenetic relatedness. The capsid gene (cp) was optimised for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N. benthamiana). Virus-like particles (VLPs) were purified and analysed using transmission electron microscopy. Virions from a Palm cockatoo (Probosciger aterrimus) were purified and a BFDV dsDNA molecular clone was synthesised and replication assessed in 293TT mammalian cells and N. benthamiana using rolling circle replication and qPCR. Two distinct BFDV phylogenetic clusters were reported for Cape parrots, and a direct correlation was seen between viral load in the blood and clinical signs in PBFD-afflicted birds. The CP was successfully expressed in N. benthamiana, and increased through optimisation of Agrobacterium infiltration density and the inclusion of the NSs silencing suppressor. The CP formed VLPs, which were shown to be morphologically similar to infectious virions. The dsDNA molecular clone was shown to replicate autonomously in mammalian 293TT cells, and in plants with the assistance of the Bean yellow dwarf virus replication associated protein (Rep). BFDV genetic diversity in Cape parrots highlights the importance of ensuring new strains are not inadvertently introduced into the wild. This is the first systematic investigation of virus diversity in Cape parrots and assessment of BFDV viral load in a wild psittacine population. The CP was successfully produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is the first reported replication of BFDV in tissue culture, and will greatly expand the scope of available research.
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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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    Enhancement of plant expression vectors using replication and silencing suppressor elements
    (2018) Jacobs, Raygaana; Hitzeroth, Inga; Rybicki, Edward; Regnard, Guy
    Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines.
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    Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum
    (BioMed Central Ltd, 2007) Kohl, Thomas; Hitzeroth, Inga; Christensen, Neil; Rybicki, Edward
    BACKGROUND:We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. RESULTS: Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 mug/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 mug/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with ~50 mug of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro. CONCLUSION: We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.
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    Expression optimization of a human papillomavirus type 16 therapeutic vaccine candidate in Nicotiana benthamiana leaves
    (2016) Do Rosario Yanez, Romana de Jesus; Hitzeroth, Inga; Rybicki, Edward P; Lamprecht, Renate
    High risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. The three approved prophylactic vaccines do not benefit already infected individuals; therefore, there is still an urgent need for therapeutic vaccines. The HPV oncoproteins E6 and E7 are ideal targets for the development of such vaccines, as they are expressed throughout the viral life cycle and in tumours. They could be used to elicit strong cytotoxic lymphocyte (CTL) responses which would aid in viral clearance, and could also be effective against tumours. Granadillo et al. (2011) developed an Escherichia coli-produced therapeutic vaccine candidate, consisting of the HPV-16 E7 protein and a cell membrane- penetrating and immunomodulatory peptide (LALF), whose fusion to HPV-16 E7 aided in the immunogenicity and antigen presentation of the oncoprotein. However, such vaccines need not only to be effective, but also to have a low cost. Plant expression systems represent an attractive alternative to conventional expression systems based on bacterial, yeast, mammalian and other cell cultures, and are potentially far more cost- effective. The aim of the present project was to produce LALF-E7 in Nicotiana benthamiana leaves via Agrobacterium-mediated transient expression, and to optimize its expression, extraction and purification. This was done by expressing LALF-E7 using different expression vectors, testing different subcellular localizations, and testing the effect of gene silencing suppressors. By using our group's replicating expression vector and targeting LALF-E7 to the chloroplasts, the expression of the candidate vaccine in N. benthamiana leaves was increased 26.8 fold compared to non-replicating vectors or cytoplasmic localization. Furthermore, silencing suppressors did not significantly increase the expression of LALF-E7 when expressed by the replicating vector and targeted to the chloroplasts. I showed, by fluorescence microscopy, that LALE-E7 was indeed being targeted to the plants' chloroplasts and that it possibly forms proteins bodies (PBs) that are closely associated to the chloroplast envelope. I further hypothesized a mechanism by which the PBs-like structures form. Once the expression of LALF-E7 was optimized in plant leaves, a purification strategy was developed by testing different extraction methods and using metal ion affinity chromatography. The extraction protocol developed pre-purified LALF-E7 by removing the majority of soluble proteins from the final extract. However, LALF-E7 was not fully purified by affinity chromatography, suggesting that other purification strategies should be used. Finally, I tested the partially purified plant-produced LALF-E7 candidate, and compared it to the E. coli-produced counterpart, in tumour regression experiments using mice as animal models. Due to low antigen doses and a large number tumourigenic cells used to inoculate the mice animal models, the effect of the plant-produced LALF-E7 as a therapeutic vaccine was inconclusive. However, it was suggested that it could potentially be comparable to the E. coli-produced counterpart. In summary, I report for the first time the entire chain of research involving the expression of LALF-E7 in plants, its extraction, purification and the testing of its immunogenicity in a mouse model. This research also suggests new avenues for the use of the LALF peptide as a PB-inducer which could be useful in increasing the expression of other recombinant proteins.
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    HPV pseudovirion production in plants
    (2013) Kennedy, Paul; Rybicki, Ed; Hitzeroth, Inga; Meyers, Ann
    Human papilloma virus (HPV) infection is the most common etiological agent of cervical cancer, the most common cancer in women in Africa. The lifecycle of HPV has historically made the virus difficult to culture in vitro, and this has hindered the study of the virus, as well as development of vaccines. The development of synthetic HPV particles, such as virus-like particles (VLPs) and more recently pseudovirions (PsVs), has allowed for unprecedented insights into the lifecycle and immunology of this virus. This has led to the development of two currently available vaccines, namely Cervarix™ and Gardasil®. Cervarix offers protection against high-risk HPV types 16 and 18, while Gardasil offers further protection against types 6 and 11. Both of these vaccines are based on major capsid protein L1 Virus-like particles (VLPs). While these vaccines show no loss of efficacy, further work is underway to develop a second generation HPV vaccine that is cheap, stable and displays cross-neutralising activity across a broader range of HPV types. The recent efficient methods for intracellular production of HPV PsVs encapsidating nonpapillomaviral DNA (pseudogenomes) has allowed for development of a robust and sensitive pseudovirion-based neutralisation assay (PBNA), which has become the gold standard neutralisation assay for the testing of candidate HPV vaccines. The currently accepted PsV production method utilises mammalian cell culture to produce HPV PsVs, encapsidating a SEAP reporter plasmid, at high titres. While this is an effective method of PsV production, mammalian cell culture is expensive and time-consuming. Transient recombinant protein expression in plants offers a rapid and cost-effective alternative to mammalian cell culture. Here, we developed a method of high-titre HPV PsV production in plants. The autonomously replicating plant vector, pRIC3, was modified to include mammalian reporter cassettes encoding luc or SEAP, for the production of reporter pseudogenomes DNA in plants by Agrobacterium-mediated transient expression. The SEAP and luc cassettes were introduced into pRIC3 upstream of the plant cassette, which was included only to increase the final pseudogenome size for efficient packaging into PsVs. The SEAP cassette was also introduced into pRIC3 in place of the plant cassette, to form a smaller pseudogenome. Thus three vectors were created, namely pRIC3-mSEAP+ (6.4Kbp pseudogenome), pRIC3-mluc+ (7.4Kbp pseudogenome), and pRIC3-mSEAP (4.8Kbp pseudogenome), which would produce pseudogenomes that covered the full range of plasmid sizes incorporated by assembling HPV capsid proteins in vivo. All three replicating vectors demonstrated the formation of a replicon, and autonomous replication, in Nicotiana benthamiana plants. Each of these vectors were co-infiltrated with the non-replicating transient plant expression constructs pTRAc-hL1 and pTRAc-hL2, which encode human-codon optimised forms of HPV-16 major and minor capsid proteins, respectively. It was expected that encapsidation of replicon DNA as a pseudogenome into assembling HPV particles would result in the production of HPV PsVs in planta. In addition, L1 and L2 were expressed in the absence of replicon DNA to form L1/L2 VLPs. Particles were extracted from plant material at four days post-infiltration, using a modified VLP extraction protocol. HPV particles were separated on the basis of isopycnic caesium chloride density gradient ultracentrifugation, dialysed against high-salt PBS and identified by fractionation and probing with an anti-L1 antibody. Particles corresponding to the buoyant density of pseudovirions were seen in samples with or without replicon DNA. Western blotting showed that all particles had incorporated both L1 and L2 proteins. Particles were digested with proteinase K to release encapsidated pseudogenome DNA and PCR confirmed the presence of replicon-specific DNA in each PsV. Electron microscopy confirmed the presence of HPV-16 PsVs in all samples. To test whether plant-produced HPV-16 PsVs could be used in pseudovirion-based neutralisation assays, mammalian cells were pseudoinfected with purified mSEAP, mSEAP+ or mluc+ PsVs. mSEAP and mluc+ PsVs elicited a reporter gene response in mammalian cells 72 hours post-infection using SEAP and luciferase assays, respectively, while mSEAP+ PsVs showed no reporter gene expression in mammalian cells. PsVs incubated with a known HPV-16 neutralising antibody showed partial neutralisation of mSEAP PsVs and complete neutralisation of mluc+ PsVs To our knowledge, this is the first demonstration of production of HPV PsVs in plants, and their use in a PBNA. Further, it is the first demonstration of production of HPV L1/L2 VLPs in plants. While much work remains to improve plant production and purification methods of PsVs, as well as mammalian expression following PsV pseudoinfection, this is an important step towards a new method of PsV production.
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    Human papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa: a cross-sectional study
    (BioMed Central Ltd, 2009) Moodley, Jennifer; Constant, Deborah; Hoffman, Margaret; Salimo, Anna; Allan, Bruce; Rybicki, Ed; Hitzeroth, Inga; Williamson, Anna Lise
    BACKGROUND:Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. METHODS: A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. RESULTS: The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 - 57.7) for those that were HC2 positive and had a viral load of [less than or equal to] 181.1 RLU (the median HPV viral load), and 33.8 (95% CI 6.4 - 178.9) for those that were HC2 positive with a HPV viral load > 181.1 RLU. CONCLUSION: Women initiating ARVs have a high prevalence of abnormal Pap smears and HR-HPV. Our results underscore the need for locally relevant, rigorous screening protocols for the increasing numbers of women accessing ARV therapy so that the benefits of ARVs are not partially offset by an excess risk in cervical cancer.
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    An in depth study of human papillomavirus diversity in South African women infected with HIV
    (2009) Salimo, Anna T; Hitzeroth, Inga; Williamson, Anna-Lise; Rybicki, Ed
    Cervical cancer is the second most common cancer affecting women and in most developing countries it remains the leading cause of cancer deaths. In South Africa, more than 3 400 women succumb to the disease every year and 1 in 31 women develop cervical cancer. The causative agent for cervical cancer is the Human papillomavirus (HPV). High-risk (carcinogenic) HPV types have been linked with 99% of the incidences of cervical cancer. The most common types identified in almost 70% of cervical cancer cases worldwide are HPV 16 and 18. HPV infection is very common in young healthy women and most immunocompetent individuals can clear HPV infection. However, in immunosuppresed women, clearance by host immune system is impaired. In addition, multiple HPV infections are quite common in women with Human immunodeficiency virus (HIV) infections. The objectives of this study were to identify HPV types in South African women who also had HIV infection, and secondarily, to determine if recombination of HPV genomes occurs. Determining the HPV types circulating in this country is important to enable identification of most common HPV types, in order to guide the development of vaccines against HPV infection. HPV genotyping was performed by the commercial Roche Linear Array HPV Genotyping Test.
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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.
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    Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits
    (BioMed Central Ltd, 2012) Meiring, Tracy; Salimo, Anna; Coetzee, Beatrix; Maree, Hans; Moodley, Jennifer; Hitzeroth, Inga; Freeborough, Michael-John; Rybicki, Ed; Williamson, Anna-Lise
    BACKGROUND: Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV+women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. METHODS: Total DNA was isolated from 109 cervical specimens from South African HIV+women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. RESULTS: Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively. CONCLUSIONS: Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.
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    Novel Production of Bovine Papillomavirus Pseudovirions in Tobacco Plants
    (2020-11-28) Pietersen, Inge; van Zyl, Albertha; Rybicki, Edward; Hitzeroth, Inga
    Vaccine efficacy requires the production of neutralising antibodies which offer protection against the native virus. The current gold standard for determining the presence of neutralising antibodies is the pseudovirion-based neutralisation assay (PBNA). PBNAs utilise pseudovirions (PsVs), structures which mimic native virus capsids, but contain non-viral nucleic material. PsVs are currently produced in expensive cell culture systems, which limits their production, yet plant expression systems may offer cheaper, safer alternatives. Our aim was to determine whether plants could be used for the production of functional PsVs of bovine papillomavirus 1 (BPV1), an important causative agent of economically damaging bovine papillomas in cattle and equine sarcoids in horses and wild equids. BPV1 capsid proteins, L1 and L2, and a self-replicating reporter plasmid were transiently expressed in Nicotiana benthamiana to produce virus-like particles (VLPs) and PsVs. Strategies to enhance particle yields were investigated and optimised protocols were established. The PsVs’ ability to infect mammalian cells and express their encapsidated reporter genes in vitro was confirmed, and their functionality as reagents in PBNAs was demonstrated through their neutralisation by several different antibodies. This is the first report of BPV PsVs expressed in plants and demonstrates the potential for the development of therapeutic veterinary vaccines in planta.
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    Optimization and characterisation of plant produced Human Papillomavirus pseudovirions in Nicotiana benthamiana
    (2020) Adams, Ayesha; Hitzeroth, Inga; Hendrikse, Megan; Rybicki, Ed
    Human papillomavirus (HPV) is known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. There are currently three prophylactic virus-like particle (VLP) based vaccines. These vaccines, however, do not offer protection against all HPV strains and cannot act therapeutically and so further vaccine development is still needed. The burden of HPV is also highest in low-income countries for which the vaccine costs are still quite high, and therefore alternative methods of vaccine production and testing are needed. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and encapsidate up to 8Kb of pseudogenome DNA without the need of an encapsidation signal. HPV PsVs are used to test neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells; however, it has recently been shown that HPV PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. While, a current problem with plant HPV PsV production is low yields, research has shown that using pseudogenome DNAs between 5-7Kb increases yields of papillomavirus PsVs in mammalian cells. Therefore, the objective of this study was to determine the optimal pseudogenome size for encapsidation by plant produced PsVs, in order to increase the amount of PsVs in a sample as opposed to VLPs. Pseudogenome constructs encoding Enhanced Green Fluorescent Protein (EGFP )and ranging in size from 4.8Kb – 7.8kb were cloned into a geminivirus-derived replicating vector, transformed into Agrobacterium tumefaciens and then infiltrated into Nicotiana benthamiana along with plant expression vectors encoding the HPV 35 L1 and L2 capsid proteins. Particles were purified by iodixanol density gradient ultracentrifugation and the 27% and 33% fractions of this gradient analysed. Transmission electron microscopy (TEM) was used to confirm particle assembly and L1 expression was quantified by ELISA. Particles were disrupted with proteinase K and quantitative PCR was used to quantify the encapsidated DNA. Ratios of encapsidated DNA to L1 capsid protein were calculated for each of the PsV samples with different sized pseudogenomes, to account for batch-to-batch variation and as an approximation of which size pseudogenome is better encapsidated. Infective ability of the particles was analysed by incubating the PsVs onto HEK293TT cells and then checking for DNA delivery and protein expression by measuring EGFP expression by Western blots. The results showed that PsVs are found predominantly in the 27% fraction of the iodixanol gradient whereas the 33% fraction of the gradient appears to only contain VLPs. The data also indicated that the smaller pseudogenomes, were packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained when the 4.8Kb pseudogenome was used, compared to when the larger 5.8 - 7.8Kb pseudogenomes were used. Thus, the results showed that smaller pseudogenomes, around 4.8Kb, should be used for the plant production of HPV 35 PsVs as they are better packaged than larger pseudogenomes and thereby produce higher yields of functional PsVs.
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    Plant production and immunogenic characterisation of Human papillomavirus chimaeric vaccines
    (2011) Pineo, Catherine; Rybicki, Ed; Hitzeroth, Inga
    Cervical cancer is primarily caused by infection with Human papillomavirus (HPV) and is a global concern, particularly in developing countries which contain ~80% of the cervical cancer burden. Current HPV L1 major capsid protein virus-like particle (VLP)-based vaccines are effective in the type-specific prevention of infection and associated disease. However, the high cost of the vaccines has limited their widespread application, and cytological screening programmes are still required to detect malignant lesions associated with the non-vaccine types, particularly in HIV-infected populations.
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    Plant production of Gonococcal peptide vaccine, candidate peptide display with HPVs
    (2024) Lindsay, Sarah; Hitzeroth, Inga; van Zyl Albertha
    Human Papillomavirus (HPV) and Neisseria gonorrhoeae (Ng) are prominent pathogens responsible for a significant proportion of the global burden attributed to sexually transmitted infections. While vaccines targeting HPV have seen success, an effective vaccine against gonorrhea remains elusive, as antimicrobial resistance continues to be a growing threat. This study explores the innovative approach of plant-based production for a dual vaccine against HPV16 and gonorrhea, utilizing N. benthamiana as the expression host. The vaccine design involves the incorporation of a gonorrhea peptide, mimicking the 2C7 epitope of the surface molecule LOS on gonorrhea, into the surface DE loop of HPV16 virus-like particles (VLPs). These chimeric VLPs as well as HPV16 VLPs were expressed and successfully purified from plants and demonstrated self-assembly into VLPs. The choice of N. benthamiana as the expression system is informed by its suitability for efficient and cost-effective recombinant protein production. Immunological evaluations were conducted in mice to assess the immune response elicited by the dual vaccine. The mice displayed robust antibody responses against both HPV and gonorrhea, indicating the insertion of the peptide does not disrupt the binding of antibodies to HPV16 and highlights the potential of the vaccine candidate to induce a dual protective immunity. To assess the protective efficacy of the vaccine candidate, a challenge model was developed and optimized, involving the use of HPV16 PsVs containing luciferase (FLuc). The challenge model was refined through a comparison of secreted and non-secreted reporter proteins, followed by comparison of plant and mammalian expression systems. This showed thatHPV16 PsVs containing non-secreted FLuc produced in mammalian cell culture were optimal for the challenge. Mice vaccinated with the plant-produced dual vaccine demonstrated protection against HPV infection upon challenge. In summary the chimeric and HPV16 VLP vaccine candidates were expressed in an Agrobacterium-mediated transient system in plants. The immunogenicity of the vaccine candidates was accessed in a mouse model and a further challenge model. The results demonstrated the potential of using VLPs in the display of foreign epitopes in the fight against gonorrhea and HPV. The findings contribute valuable insights into the development of vaccines against sexually transmitted infections, paving the way for innovative strategies in the field of VLP display molecules and plant molecular pharming.
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    Production and immunogenicity of chimaeric human papillomavirus-like particle vaccines
    (2010) Burger, Marieta; Rybicki, Ed; Hitzeroth, Inga
    Human papillomavirus (HPV) infection, specifically with oncogenic types, has been implicated in effectively all cervical cancer cases. Cervical cancer is a global health burden, especially in the developing world. Up to 18 types of HPV are considered oncogenic, of which HPV -16 and -18 cause 70% of cervical cancer cases worldwide. Two vaccines are available on the market: Gardasil(R), targeted against HPV -16, -18; -6 and -11, and Cervarix(TM), against -16 and -18. Both vaccines are based on the L1 capsid proteins of the types they are targeted to and are efficient, pro- phylactic, typespecific vaccines. However, two problems remain: they do not protect against nonvaccine types, that may cause a significant proportion of cancers specifically in African and HIV- positive populations, and they cannot be used to treat existing infections. We designed eight different chimaeric vaccines.
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    Setting up a platform for plant-based influenza virus vaccine production in South Africa
    (BioMed Central Ltd, 2012) Mortimer, Elizabeth; Maclean, James; Mbewana, Sandiswa; Buys, Amelia; Williamson, Anna-Lise; Hitzeroth, Inga; Rybicki, Edward
    BACKGROUND:During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. RESULTS: For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. CONCLUSIONS: We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.
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    The 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, Ed
    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.
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    The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent
    (2019) Verbeek, Matthew James Robert; Hitzeroth, Inga; Mbewana, Sandiswa; Rybicki, Edward
    Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genus Orthobunyavirus, family Peribunyaviridae., and order Bunyavirales. Discovered in arthropods and humans in Nigeria, it was soon identified as a possible cause for cases of neurological disease in horses within South Africa. Studies have shown South African veterinarians who had come into contact with such cases tested positive for antibodies against the virus. Therefore, SHUV is being further investigated as a potential cause of neurological disease within humans and there is a need to develop appropriate quick and effective diagnostic reagents to allow for surveillance of the virus. The main focus for this study was the development of diagnostic reagents centred around the nucleocapsid (N) protein of the SHUV. The N proteins of closely related members of the order Bunyavirales have shown to be highly abundant in infection and cause an immune response in the infected hosts thus making it the ideal target. Using available SHUV genome sequences and data, the N protein gene was designed and synthesised to be expressed in both Escherichia coli and plant expression systems. The expression of the N protein in E. coli, followed by subsequent washing with BugBuster, led to a final mass of 5.1 mg of the SHUV N protein from a 1000 ml culture. This led to a SHUV N yield of 5.1 µg/ml of culture and was measured to make up 69.5% of the total soluble protein. The immunisation of rabbits with this recombinantly expressed SHUV N allowed for the development of polyclonal antibodies which were successfully used in immunoblot studies to detect plant produced SHUV N protein. Plants are an effective and possibly cheaper alternative production system to bacterial, mammalian, or insect cell cultures and thus the N protein was transiently expressed in N. benthamiana plants using Agrobacterium tumefaciens-mediated infiltration. The recombinant protein produced underwent purification using nickel affinity chromatography. This led to yields of 2.248 mg of SHUV N protein from 35 plants which gave a yield of 9.9 mg/kg of raw plant material. This purified plant produced N protein acted as an antigen for diagnostic assays such as ELISA, which was used to screen known SHUV infected sera. This led to mixed results due to the limited sera samples available. However, as a proof of concept, it has shown great potential and thus opens the door to a possible inexpensive dual-use assay for use in the diagnoses of both animal and human SHUV infection.