OpenUCT :: Browsing by Author "Rybicki, Edward"

Browsing by Author "Rybicki, Edward"

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Open Access
Abrogation of contaminating RNA activity in HIV-1 Gag VLPs
(BioMed Central Ltd, 2011) Valley-Omar, Ziyaad; Meyers, Ann; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/mug Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.
Open Access
Adaptive evolution by recombination is not associated with increased mutation rates in Maize streak virus
(BioMed Central Ltd, 2012) Monjane, Aderito; Pande, Daniel; Lakay, Francisco; Shepherd, Dionne; van der Walt, Eric; Lefeuvre, Pierre; Lett, Jean-Michel; Varsani, Arvind; Rybicki, Edward; Martin, Darren
BACKGROUND: Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus). RESULTS: Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV and TYLCV we observed similar strand specific mutation biases arising predominantly from imbalances in the complementary mutations G->T: C->A. CONCLUSIONS: While our results suggest that recombination does not strongly influence mutation rates in MSV, they indicate that high geminivirus mutation rates are at least partially attributable to increased susceptibility of all geminivirus genomes to oxidative damage while in a single stranded state.
Open Access
Development of microalgae as a biopharming platform
(2019) Els, Johann Hendrik; Rybicki, Edward; Hitzeroth Inga; Harrison, Sue
Microalgae may be a powerful biopharmaceutical production platform that is still in its infancy of development. The research done in this project tested the feasibility of creating algal cell packs, a novel immobilised microalgae transient production platform for the expression of recombinant protein. First it had to be established whether the available plant expression vectors could be used for the transfer of genetic material into packed microalgae. The method showed successful transfer of the neomycin phosphotransferase II resistance gene (nptII). Further experiments analysed the plant expression vectors pTRAc and pRIC3.0 for expression of enhanced green fluorescent protein (EGFP) in Scenedesmus spp. by western blotting. Possible replication of the plant geminivirus-derived pRIC3.0 was then confirmed by comparing to replication in Nicotiana benthamiana by quantitative polymerase chain reaction (qPCR). Western blot results indicated EGFP expression in N. benthamiana but not in Scenedesmus. By using PCR the presence of EGFP DNA in Scenedesmus was detected but qPCR showed no increase of the pRIC3.0 replicon. Despite no detection via antibodies of EGFP in Scenedesmus, green fluorescence was observed. These initial results showed promise and points to a system that requires optimisation for increased transfection rates and protein expression. Following on from the initial work, the project set out to determine the feasibility of expressing a recombinant anti-Ebola viral inhibitor protein in three different plant based platforms namely N. benthamiana, a microalgal genus, Desmodesmus and a BY2 tobacco plant-cell culture. Protein expression was compared between the Desmodesmus algal cellpack, N. benthamiana plant expression system and BY-2 plant cell packs by western blotting. Four designs of the viral inhibitor fused to the maize ƴ-zein protein body inducing protein, ZERA, were expressed in trace quantities. Transient expression was more prominent in the algal cell packs than in N. benthamiana and BY-2 cells. The algal cell pack system may potentially be a powerful tool to test recombinant protein expression in a range of microalgal hosts via Agrobacterium-mediated genetic transfection. The future development of recombinant protein expression platforms could be enhanced by rapid testing of protein production in different species. Refinement needs to be done on the algal cell pack to increase transfection efficiency and expression in microalgae to produce commercially viable quantities of heterologous protein.
Open Access
Engineering the Nicotiana benthamiana secretory pathway for the production of Lujo virus vaccines and diagnostic tools
(2023) Isaacs, Abdul; Rybicki, Edward; Meyers Ann Elizabeth
Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccination offers a solution to reduce the difficulties that accompany viral outbreaks. Lujo virus, an emerging arenavirus responsible for causing a devastating haemorrhagic fever with an 80% mortality rate, currently has no vaccines or diagnostic tools available. In this study we developed a production pipeline in HEK293T cells and N. benthamiana for the protein LUJV GP-C∆TM (Lujo virus glycoprotein precursor without the transmembrane region). The LUJV GP-C∆TM was constructed from gene sequences of the envelope glycoprotein of Lujo virus and then adapted to production in HEK293T cells using the DNA expression vector pTHpCapR. An N. benthamiana plant protein expression system was developed in parallel to compare the production utility of both systems with an emphasis being placed on the plant system. Plant expression systems are arguably cheaper and more easily automatable than traditional mammalian expression technologies. This makes them suitable for vaccine protein production in lower socio-economic countries that have an overwhelming burden of disease and poor health care systems. LUJV GP-C∆TM was successfully expressed in both HEK293T cells and N. benthamiana. It was confirmed that the LUJV GP-C∆TM undergoes the post translational modifications glycosylation and proteolytic cleavage in recombinant HEK293T cells and was produced in a conformation that allowed successful purification via a His tag sequence that was inserted into the LUJV GP-C∆TM protein gene sequence. On the other hand, N. benthamiana does not endogenously express the Site-1 protease needed for cleavage of the Lujo glycoprotein, and thus this needed to be co-expressed. Attempts were made to detect the Site-1 protease including extraction into buffers with different pHs and ammonium sulfate precipitation to concentrate the protein. However, I was not able elicit proteolytic cleavage of the LUJV GP-C∆TM nor detect the Site-1 protease in N. benthamiana. This is probably attributable to the innate responses against the hostile nature of proteases to non-native expression hosts. Proteins were expressed in N. benthamiana through the use of previously established protocols utilizing recombinant Agrobacterium tumefaciens to deliver synthesized genes to plant cells and induce their expression. It was determined that co-expression of the molecular folding chaperone calreticulin is necessary for LUJV GP-C∆TM to accumulate at detectable levels. Co-expression of an oligossacharyl transferase LmSTT3D, isolated from Leishmania major, may increase the glycan occupancy of the LUJV GP-C∆TM protein, indicated by a molecular mass shift. However, further experimental lines of evidence are needed -such as glycosylation mapping to determine if this is the case. Other parameters such as the optimal day of protein harvest and optical density of recombinant Agrobacterium tumefaciens strains were recorded. Collectively these findings serve as a prototype pipeline for the production of commercially relevant immunogens, diagnostic tools and virus-like particles. This study narrows the focus for bottlenecks in plant protein production, not just for Lujo virus proteins, but for arenaviruses generally and potentially also other haemorrhagic fever-causing viruses. Future efforts should be directed towards addressing the barriers to plant production of complex viral antigens, and to further investigate the utility of mammalian cell-produced LUJV GPC∆TM in animal studies.
Open Access
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.
Open Access
Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts
(BioMed Central Ltd, 2009) Harkins, Gordon; Delport, Wayne; Duffy, Siobain; Wood, Natasha; Monjane, Aderito; Owor, Betty; Donaldson, Lara; Saumtally, Salem; Triton, Guy; Briddon, Rob; Shepherd, Dionne; Rybicki, Edward; Martin, Darren; Varsani, Arvind
BACKGROUND:Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. RESULTS: We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 x 10-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Reunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. CONCLUSION: The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.
Open Access
Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias
(BioMed Central Ltd, 2008) van der Walt, Eric; Martin, Darren; Varsani, Arvind; Polston, Jane; Rybicki, Edward
BACKGROUND: Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered. RESULTS: We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 x 10-4 and 7.9 x 10-4 subs/site/year. CONCLUSION: These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.
Open Access
Expression of HIV-1 antigens in plants as potential subunit vaccines
(BioMed Central Ltd, 2008) Meyers, Ann; Chakauya, Ereck; Shephard, Enid; Tanzer, Fiona; Maclean, James; Lynch, Alisson; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. RESULTS: Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. CONCLUSION: Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.
Open Access
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.
Open Access
A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family
(BioMed Central Ltd, 2009) Varsani, Arvind; Shepherd, Dionne; Dent, Kyle; Monjane, Aderito; Rybicki, Edward; Martin, Darren
BACKGROUND:We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera. RESULTS: Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. CONCLUSION: Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages.
Open Access
More men than women make mucosal IgA antibodies to Human papillomavirus type 16 (HPV-16) and HPV-18: a study of oral HPV and oral HPV antibodies in a normal healthy population
(BioMed Central Ltd, 2006) Marais, Dianne; Sampson, Candice; Jeftha, Anthea; Dhaya, Dherendra; Passmore, Jo-Ann; Denny, Lynette; Rybicki, Edward; Van Der Walt, Eric; Stephen, Lawrence; Williamson, Anna-Lise
BACKGROUND:We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. METHODS: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. RESULTS: Oral HPV infection was highest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09-12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97-6.2, P = 0.04). CONCLUSION: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation.
Open Access
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.
Open Access
The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine
(BioMed Central Ltd, 2011) Tanzer, Fiona; Shephard, Enid; Palmer, Kenneth; Burger, Marieta; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND:One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. RESULTS: The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. CONCLUSIONS: We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.
Open Access
Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution
(BioMed Central Ltd, 2011) Monjane, Aderito; van der Walt, Eric; Varsani, Arvind; Rybicki, Edward; Martin, Darren
BACKGROUND:Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. RESULTS: Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. CONCLUSIONS: The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.
Open Access
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.
Open Access
Short-term dynamics of nano- and picoplankton in the southern Benguela upwelling system
(2022) Dames, Nicole Rebecca; Moloney, Coleen L; Rocke, Emma; Rybicki, Edward; Pfaff, Maya
Wind driven coastal upwelling influences the overall physical and chemical properties of coastal regions, as well as the small phytoplankton and microbial communities responsible for the productivity and biogeochemistry governing many of these properties. These environmental changes can influence picoplankton (0.3–3 µm) and nano-picoplankton (0.3–10 µm) at different time scales; in this thesis daily changes were of interest because of the cyclic (3–7 days) nature of wind-driven upwelling. Daily variability of picoplankton was studied during an upwelling cycle at a single station in Elands Bay. Using amplicon sequencing of the 16S and 18S rRNA gene region, as well as additional supplementary environmental data, it was found that picoplankton diversity, community structure and primary metabolism varied between the active and relaxation periods of an upwelling cycle. The results highlighted the complexity of picoplankton dynamics in variable environmental settings. However, the question then became whether nano-picoplankton dynamics were as complex in a post-upwelling setting. This was assessed in autumn (post-upwelling period) in St. Helena Bay by measuring primary productivity and nitrogen cycling over five days from three depths at a single station. Using stable isotope tracer and flow cytometry analyses it was determined that primary productivity was supported by regenerated production and that nano-picoplankton were responsible for up to 90% of the net primary production, with nanoeukaryotes and heterotrophic bacteria dominating at the surface and at depth. Increased resolution of nano-picoplankton community composition, structure and potential metabolism was obtained using metagenomic analyses of samples taken at the same depths and days as the productivity study. A strong depth-differentiation in community structure and potential metabolism was found over the five-day period, with little variability observed from day to day. Metagenome abundances of transporter genes for processes like ammonium uptake and nitrite oxidation were found to be good indicators of measured process rates using isotope tracers. This research has highlighted the complex structure of picoplankton and nano-picoplankton communities in a coastal setting, and has shown how diversity, function and biotic interactions are strongly influenced by the properties of the surrounding water column.
Open Access
Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media
(BioMed Central Ltd, 2012) Lynch, Alisson; Meyers, Ann; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND:HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability.FINDINGS:We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4degreesC, -20degreesC and 70degreesC) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at 70degreesC retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. CONCLUSIONS: Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at 70degreesC for 12 months is most effective in retaining VLP stability.
Open Access
The characterisation of a novel Xanthomonas Bacteriophage
(1999) Petersen, Yolanda; Qhobela, Molapo; Rybicki, Edward
During the 1994-95 growing season, an apparently new disease was observed on Brassica oleracea var. capitata (cabbage) seedlings in nurseries of the province of KwaZulu-Natal, South Africa. Named chocolate spot disease of crucifers because of the characteristic symptoms of dark brown to black lesions with transparent centers, this disease was particularly severe on the more mature leaves of stressed seedlings and resulted in serious economic losses. The causal agent, determined to be a strain variant of Xanthomonas campestris pv. campestris was found to be associated with bacteriophage-like particles. The optimal growth conditions for production of the bacteriophage-like particles by the chocolate spot pathogen were determined and a purification protocol developed that yielded particles in a quantity and quality adequate for further analysis. Based on a comparison of the particle and bacterial protein reactions with the antiserum raised against the purified particles, it was concluded that the particles were not vesicles originating from the bacterial membrane, but were therefore most probably of bacteriophage origin. Physicochemical characterisation showed that the putative phage is a tailless isometric particle with a diameter of 33.45 nm, sedimentation coefficient of 85S and a density in CsCl of 1.347 g/ml. The phage is relatively stable with respect to pH, solvents and at temperatures of 40°C and 60°C. The particles contain 3 major proteins of 94, 32 and 21.8 kDa as well as two minor proteins of 40 and 25.7 kDa, and it has a single-stranded DNA genome. Biological characterisation of the novel bacteriophage indicates that it is a temperate phage which does not cause visible plaque formation on solid media and is not inducible by the external factors, mitomycin C and ultraviolet radiation. Serological tests showed that the phage is present in all isolates of the chocolate spot pathogen and that similar particles are associated with the X. campestris pathovars aberrans, armoraciae, campestris and raphani. No serological relationship was detected between the novel phage and the X. c. pv. campestris phage, RR68. However, the novel phage antibodies recognised 3 proteins of molecular weights 21.8, 13.79 and 12.64 kDa in the X. c. pv. campestris phage, HT3h. These proteins are localised in the phage capsid. Although phage-like particles were detected in the novel phage host PCB 22 following electron microscopy of ultra-thin sections, immunogoldlabelling could not confirm whether the particles were of phage origin. The single-stranded DNA genome of the phage hybridised with a 53.9 kb extrachromosomal DNA element. Since its size precludes this element being packaged as single-stranded DNA into a capsid 33.45 nm in diameter, it is most probable that this 53.9 kb DNA element is an indigenous plasmid into which the double-stranded form of the phage genome has integrated. However, this could not be confirmed by the results of the nucleic acid hybridisation tests. The 53.9 kb extrachromosomal element was cloned and several recombinant plasmids sequenced. No typical phage genes were identified. However, fragments of a Xanthomonas avirulence (avr) gene interrupted by part of a transposon sequence were identified. A 201- 262 base region of clone pSSI shared a 98% identity to the 3' end of a group of Xanthomonas avr genes, while 141-311 bases had a high degree of nucleotide similarity to the 5' end of the avr genes. These two regions of avr gene similarity which appear to converge, are interrupted by 1259 bases sharing 99% nucleotide identity to the 4864-6126 bp position of the 6938 bp X. campestris transposon, ISXC5 and 298 bases with 97% similarity to the 6644-6938 bp region of the transposon. The 1259 and 298 base regions are in tum separated by an unsequenced region of 518 bases. The presence of the avr and transposon sequences (usually located on plasmids or the bacterial chromosome) on the extrachromosomal element strengthens the hypothesis that the phage genome is integrated into an indigenous plasmid.
Open Access
The design, development and characterization of a self-replicating DNA expression technology
(2020) de Moor, Warren Ralph Josephus; Rybicki, Edward; Regnard, Guy; Williamson, Anna-Lise
High quality T-cell immunogenicity can be an elusive type of immunity to generate and one that is often sought after by virologists, immunologists and cancer researchers alike. When T-cell immunity is generated using current methodologies the quality and magnitude of the immunological response achieved is often weak and unable to create protective immunity. Among current methods, DNA vaccines, generate highly specific T-cell immunity towards targeted antigens, and do not suffer from issues like misdirected vector targeted immunity, like viral based vectors. DNA vaccines, however, face a variety of their own weaknesses. These include, inefficient delivery, high biological loss inside the body, and the inability to counteract or avoid immediate innate cellular defence mechanisms, which limit their ability to persist inside a host cell. For these reasons, DNA vaccines are usually combined with more conventional viral vaccines in what is known as a DNA prime and viral boost regiment strategy. Combining them works well and results in improved immunity towards targeted antigens that is superior to what is obtained when either DNA or recombinant vaccines are used alone. To address many of the core issues faced by DNA vaccines, I report here on the design, development and characterization of a self-replication DNA gene expression technology. This novel DNA expression system employs a form of DNA replication (known as rolling circle replication) to generate a self-replicating DNA amplicon that can amplify its own copy number and the relative localised levels of antigen expression inside transfected mammalian cells in tissue culture and within Balb/cJ mice. These capabilities help effectively mitigate many of the core issues faced by DNA vaccines. The technology developed was shown to significantly increase gene expression for eGFP and Luciferase reporter genes, with an overall average increase in expression of approximately two-fold by 48 h post transfection in HeLa S3 cells. More specifically, an increase of at least two-fold in the absolute maximum level of the gene of interest per cell was also observed. Such localised doubling in antigen expression, at the cellular level, is believed to enhance innate immune activation and improve the overall immune response. Experimental results indicated that gene expression levels by this technology is non static in nature and appears to increase in magnitude within affected cells over time as was hypothesised. This provided strong evidence that the replication technology appears to be functioning as was expected and was able to demonstrate the ability to elevate antigen expression over time, potentially starting from extremely low and otherwise ineffective starting concentrations. This ability has potential to effectively mitigate many of the issues associated DNA vaccines such as low and ineffective delivery. This capability was observed in tissue culture as a steady increase in reporter gene expression levels across the entire range of DNA transfection levels. Furthermore, the increases in gene expression were observed to continue to amplify over time, eliminating the presence of weakly fluorescing cells in tissue culture. By 11 days post transfection, every observable cell transfected with the replication expression system, was observed to have extremely high levels of fluorescence. With recorded fluorescence levels being as bright or brighter than the highest levels obtained under normal transfections with no replicative plasmids (~48-72 h). Unique cellular responses to the presence of the replicating gene expression technology were also observed. These included an apparent slowdown in cellular metabolic activity and growth among cells transfected with replicating vectors. This was observed as a decrease in cellular division and total cell number by ~50%, by 48 h post transfection. This was accompanied by significant increases in cell size, internal cellular granularity, and gene-of-interest expression per cell. These changes were observed among all cells regardless of their relative DNA transfection level. This was demonstrated by assessment of the change in the range, mean, median, skewness and standard deviation of the cellular distribution curves for eGFP expression, cell size and internal cellular granularity. These observations provided further evidence of the dynamically changing and active nature of this technology. This also provided evidence that the replicating gene expression technology has a definitively different kind of cellular impact and effect on transfected cells compared to non-replicating DNA expression systems. Pilot studies to test the technology in Balb/cJ mice indicated, the technology appears to be functional within this animal model and was able to increase gene of interest (eGFP) expression levels compared to an equivalent non-replicating DNA expression vector control. Furthermore, these animal experiments also demonstrated significant increases in the maximum possible level of expression achieved within localised ‘hot spots' of muscle fibre bundles. This effect appeared to increase following transient addition of additional replication associated protein (Rep), giving further evidence this technology appears to be functional within the Balb/cJ animal model. Suggesting that the rate at which the replication amplification process occurs, may also be manipulated by adjusting Rep concentration. Finally, an antiviral response gene array was run to look for evidence that the replicating gene expression technology could increases antiviral response gene activation, to possibly improve T-cell activation and immunity. The array provided evidence improved antiviral response gene activation was occurring however the data was inconclusive in nature and further investigation is needed to verify these preliminary findings. The array also showed significant evidence of Rep induced Caspase 10 (CASP10), gene suppression. This suggests that Rep may play a role in the survival and virulence ofBFDV by acting as a suppressor of cellular apoptosis in a concentration-dependant manner and is worth investigating further.
Open Access
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.