OpenUCT :: Browsing by Author "Rybicki, Edward P"

Browsing by Author "Rybicki, Edward P"

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A deletion and point mutation study of the human papillomavirus type 16 major capsid gene
(2006) Varsani, Arvind; Williamson, Anna-Lise; Jaffer, Mohamed A; Rybicki, Edward P
Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A→T mutation at residue 266 (A266T), and of a C→G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428–483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2–9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428–465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.
Open Access
Characterisation of two aphid picorna-like viruses
(1988) Williamson, Carolyn; Rybicki, Edward P; Von Wechmar, M Barbara
A new aphid virus, aphid lethal paralysis virus (ALPV), was isolated from laboratory-propagated Rhopalosiphum padi aphids co-infected with R. padi virus (RhPV). ALPV and RhPV were separated and ALPV was characterised in detail. Virions are isometric with a diameter of 26 nm, a sedimentation coefficient of 164 Sand a density in CsCl of 1.34 g/ml. Virions contain a 9.7 kb polyadenylated, singlestranded RNA and three major proteins with molecular weights of approximately 30 kilodaltons. By characterising RhPV further, two additional putative capsid proteins were found, an RNA poly(A) tract was detected and an RNA size of 10 kb was determined. A South African isolate of RhPV (RhPVoFs) was found to be serologically identical but physically distinct from a USA isolate. Complementary DNA was synthesized from RhPVOFS RNA and cloned into the plasmid vector, pBR322. This clone was used for the detettion of virus in aphids. ALPV and RhPV are serologically unrelated. ALPV is serologically distantly related to two insect picornaviruses, cricket paralysis virus (CrPV) and Drosophila C virus. No nucleic acid homology was detected between ALPV cDNA and CrPV by dot-blot hybridization. ALPV is serologically unrelated to seven other insect picornalike viruses. RhPV is serologically unrelated to any of the above mentioned viruses. ALPV and RhPV RNAs were efficiently translated in rabbit reticulocyte lysate into high molecular weight polypeptides, the sum of which exceeded the coding capacity of the genomes. Putative capsid precursor proteins of ALPV and RhPV were identified by immunoprecipitation. ALPV translation products were post-translationally cleaved as demonstrated in pulse-chase experiments and in experiments using a translation inhibitor. The efficiency of cleavage was concentration-dependent indicating the action of a protease. In parallel experiments with RhPV RNA, no evidence of post-translational cleavage was observed. In a survey of aphids collected in South Africa, ALPV and RhPV were detected in aphids from two major small-grain producing areas. Both viruses were found to naturally infect most of the cereal aphid species found in this country. ALPV and RhPV infections of R. padi resulted in a marked reduction in longevity and fecundity relative to uninfected aphids. Both viruses were found to be horizontally and vertically transmitted through aphid populations, and aphid host plants and aphid predators could be implicated in virus dissemination. ALPV and RhPV have many properties in common with each other as well as with insect and mammalian picornaviruses. Based on this data, it is proposed that ALPV and RhPV be classified into the picornavirus group (family Picornaviridae).
Open Access
Characterization of a Novel Chimeric Theileria parva p67 Antigen Which Incorporates into Virus-like Particles and Is Highly Immunogenic in Mice
(2022-01-28) Whittle, Leah; Chapman, Ros; van Diepen, Michiel; Rybicki, Edward P; Williamson, Anna-Lise
The current method to protect cattle against East Coast Fever (ECF) involves the use of live Theileria parva sporozoites. Although this provides immunity, using live parasites has many disadvantages, such as contributing to the spread of ECF. Subunit vaccines based on the sporozoite surface protein p67 have been investigated as a replacement for the current method. In this study, two DNA vaccines expressing recombinant forms of p67 designed to display on retrovirus-like particles were constructed with the aim of improving immunogenicity. The native leader sequence was replaced with the human tissue plasminogen activator leader in both vaccines. The full-length p67 gene was included in the first DNA vaccine (p67); in the second, the transmembrane domain and cytoplasmic tail were replaced with those of an influenza A virus hemagglutinin 5 (p67HA). Immunofluorescent staining of fixed and live transfected mammalian cells showed that both p67 and p67HA were successfully expressed, and p67HA localised on the cell surface. Furthermore, p67HA was displayed on the surface of both bovine leukaemia virus (BLV) Gag and HIV-1 Gag virus-like particles (VLPs) made in the same cells. Mice vaccinated with DNA vaccines expressing p67 and p67HA alone, or p67HA with BLV or HIV-1 Gag, developed high titres of p67 and BLV Gag-binding antibodies. Here we show that it is possible to integrate a form of p67 containing all known antigenic domains into VLPs. This p67HA–VLP combination has the potential to be incorporated into a vaccine against ECF, as a DNA vaccine or as other vaccine platforms.
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Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles.
(Elsevier, 2008) Halsey, Richard J; Tanzer, Fiona L; Meyers, Ann; Pillay, Sirika; Lynch, Alisson; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward P
HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines.
Open Access
Design and production of a candidate universal influenza A vaccine in Nicotiana benthamiana plants
(2017) De Figueiredo Pinto Gomes Pera, Francisco; Hitzeroth, Inga I; Rybicki, Edward P
The influenza A virus is responsible for 250,000 to 500,000 deaths every year worldwide and millions more could die in the event of a serious pandemic. Vaccines against influenza have existed for long, but until today they have been limited by extensive production times and reduced cross-protection between different strains of the virus. This leads to a recurrent need to update the vaccine composition every year, which is both costly and inadequate to fight pandemics. An innovative approach that could improve the vaccine efficacy has been recently developed based on the selection of conserved influenza epitopes with potential to induce broader immune responses. The 23-amino acid extracellular domain of the M2 protein (M2e) is highly conserved among different influenza A strains and thus it seems like an ideal candidate for a universal influenza vaccine. However, due to its small size, it is a poor immunogen when used on its own. The aim of this project was to produce M2e-presenting virus-like particles (VLPs) in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Plants are increasingly being examined as alternative recombinant protein expression systems due to their safety, scalability and rapid production times. Moreover, numerous studies suggest the use of recombinant virus-like particles (VLPs) to increase the immunogenicity of antigens. Therefore, to obtain VLPs presenting the M2e epitope, I genetically engineered several different M2e-HA fusion proteins by replacing the hemagglutinin (HA) globular head and main epitope with five tandem repeats of M2e epitope sequences (5xM2e) from human, swine, and avian origin influenza A viruses. To increase the chances of obtaining VLPs, M2e-HA fusions either contained the HA stalk domain (5xM2e-HAstalk) or simply the transmembrane region (5xM2e-HAtrans). Furthermore, the tetramerizing leucine zipper derived from the General Control Protein (GCN4) was also included in some of the constructs to promote particle formation. In total, six different M2e-HA fusions were created: 5xM2e-GCN4-HAstalk, 5xM2e-GCN4-HAtrans, 5xM2e-HAstalk, 5xM2e-HAtrans, 1xM2e-HAstalk and 1xM2e-HAtrans. The expression of these proteins was optimized in plants by testing different conditions and using three different expression vectors. Overall, I was able to show expression after only 3 days post-infiltration for most of the M2e-HA v fusion proteins utilizing the pEAQ-HT and pRIC 3.0 expression vectors whereas expression levels with pTRAc were low or non-detectable. Once the expression of the M2e-HA fusions was optimized, the two proteins with the highest potential to form VLPs were selected for further characterization (5xM2e-HAstalk and 5xM2eHAtrans). Using transmission electron microscopy to analyse purified proteins, both 5xM2eHAstalk and 5xM2e-HAtrans were shown to assemble into VLPs resembling the shape and size of native HA VLPs. These VLPs could also be observed in the apoplastic fractions of infiltrated leaves. However, due to the low number of particles observed, the successful incorporation of the M2e peptide on the surface of the particles was inconclusive, as shown by M2e-specific immuno-gold labelling experiments. Furthermore, contrarily to previous studies, co-expression of the M2e-HA fusions with the M1 protein resulted in a decrease in recombinant protein accumulation and VLP formation in our plant system. A possible inhibition mechanism by the M1 protein is discussed. In summary, this research provides preliminary data to produce universal influenza vaccines in plants. I report here for the first time that M2e fused to either the stalk or transmembrane domain of the HA protein, can self-assemble into VLPs without any other proteins, in N. benthamiana plants. Future work on the immunogenicity of the VLPs produced in this study is required to confirm their potential as a universal influenza vaccine that can be rapidly produced.
Open Access
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.
Open Access
Development of plant-produced Bluetongue virus vaccines
(2014) van Zyl, Albertha R; Meyers, Ann; Rybicki, Edward P
Bluetongue is a disease of domestic and wild ruminants caused by Bluetongue virus (BTV). It has caused several serious outbreaks, the most recent occurring in Northern Europe in 2006 during which high mortality rates of livestock were reported. The only vaccines currently approved and commercially available for use are live-attenuated or inactivated virus strains and although these are effective, there is the risk of reversion in the case of live-attenuated strains to more virulent forms by recombination. Another drawback associated with the use of live-attenuated virus vaccines is that they are not DIVA (differentiate infected from vaccinated animals) compliant, this means that naturally infected animals cannot be distinguished from vaccinated animals. Recombinantly produced vaccines would be preferable to minimize the risks associated with live-attenuated virus vaccines and also enable the development of candidate vaccines that are DIVA-compliant. A number of recombinant vaccine candidates have been developed against BTV, with the most promising vaccine consisting of BTV virus-like particles (VLPs). BTV VLPs were successfully produced in insect cells by the co-expression of the four BTV capsid proteins (VP2, VP3, VP5 and VP7). Sheep vaccinated with insect cell-produced BTV VLPs were shown to be protected against challenge with wild type virus. However, the high costs associated with the production and scale-up of BTV VLPs in insect cells has possibly limited their widespread application. Plants – such as N. benthamiana – provides a safe, efficient and cost effective system for the production of recombinant proteins. In this study the best plant expression vector with which to co-express the four BTV serotype 8 (BTV-8) VPs – which direct formation of BTV-8 VLPs – was identified. Expression and purification of the BTV-8 VLPs was optimised with the aim of producing a VLP-based vaccine for BTV-8. It was further undertaken to develop two novel second generation plant-produced protein body (PB) vaccines that are DIVA compliant. Mice were immunised with the plantproduced VLP and PB vaccines in order to analyse their ability to elicit humoral immune responses.
Open Access
Development of plant-produced protein body vaccine candidates for bluetongue virus
(BioMed Central, 2017-05-30) van Zyl, Albertha R; Meyers, Ann E; Rybicki, Edward P
Background: Bluetongue is a disease of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), which have caused serious outbreaks worldwide. Commercially available vaccines are live-attenuated or inactivated virus strains: these are effective, but there is the risk of reversion to virulence or reassortment with circulating strains for live virus, and residual live virus for the inactivated vaccines. The live-attenuated virus vaccines are not able to distinguish naturally infected animals from vaccinated animals (DIVA compliant). Recombinant vaccines are preferable to minimize the risks associated with these vaccines, and would also enable the development of candidate vaccines that are DIVA-compliant. Results: In this study, two novel protein body (PB) plant-produced vaccines were developed, Zera®-VP2ep and Zera®-VP2. Zera®-VP2ep contained B-cell epitope sequences of multiple BTV serotypes and Zera®-VP2 contained the full-length BTV-8 VP2 codon-optimised sequence. In addition to fulfilling the DIVA requirement, Zera®-VP2ep was aimed at being multivalent with the ability to stimulate an immune response to several BTV serotypes. Both these candidate vaccines were successfully made in N. benthamiana via transient Agrobacterium-mediated expression, and in situ TEM analysis showed that the expressed proteins accumulated within the cytoplasm of plant cells in dense membrane-defined PBs. The peptide sequences included in Zera®-VP2ep contained epitopes that bound antibodies produced against native VP2. Preliminary murine immunogenicity studies showed that the PB vaccine candidates elicited anti-VP2 immune responses in mice without the use of adjuvant. Conclusions: These proof of concept results demonstrate that Zera®-VP2ep and Zera®-VP2 have potential as BTV vaccines and their development should be further investigated.
Open Access
Development of Rift Valley fever virus candidate vaccines and reagents produced in Nicotiana benthamiana
(2017) Mbewana, Sandiswa; Rybicki, Edward P; Meyers, Ann
Rift Valley fever (RVF) is a haemorrhagic fever agent caused by an infection with an enveloped negative-stranded RNA Rift Valley fever virus (RVFV). It belongs to the genus Phlebovirus in the family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing high numbers of neonatal fatalities in animals and occasional fatalities in humans. It is endemic to parts of Africa and the Arabian Peninsula, but is described as an emerging virus due to the wide range of mosquitoes that could spread the disease into non-endemic areas, posing serious health and agricultural problems. The disease can be prevented by vaccination, but there is currently no Food and Drug Administration-approved RVFV vaccine that can be used outside endemic areas, while there are two live attenuated vaccines available for use in endemic areas. These vaccines have the potential for reversion, and are therefore not recommended for use in countries where RVFV is not endemic. This indicates the need for more RVFV vaccine research and development. This work focused on the development of a RVFV vaccine candidate that would allow for differentiation between infected and vaccinated animals as well as humans.
Open Access
The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus
(1994) Hackland, Andrew F; Thomson, Jennifer Ann; Rybicki, Edward P
Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I.
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Evidence of ancient papillomavirus recombination
(Microbiology Society, 2006) Varsani, Arvind; van der Walt, Eric; Heath, Livio; Rybicki, Edward P; Williamson, Anna Lise; Martin, Darren P
An open question amongst papillomavirus taxonomists is whether recombination has featured in the evolutionary history of these viruses. Since the onset of the global AIDS epidemic, the question is somewhat less academic, because immune-compromised human immunodeficiency virus patients are often co-infected with extraordinarily diverse mixtures of human papillomavirus (HPV) types. It is expected that these conditions may facilitate the emergence of HPV recombinants, some of which might have novel pathogenic properties. Here, a range of rigorous analyses is applied to full-genome sequences of papillomaviruses to provide convincing statistical and phylogenetic evidence that evolutionarily relevant papillomavirus recombination can occur.
Open Access
The Evolutionary Value of Recombination Is Constrained by Genome Modularity
(Public Library of Science, 2005) Martin, Darren P; Walt, Eric van der; Posada, David; Rybicki, Edward P
Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.
Open Access
Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias
(BioMed Central, 2008) van der Walt, Eric; Martin, Darren P A; Varsani, Arvind; Polston, Jane E; Rybicki, Edward P
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 × 10-4 and 7.9 × 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 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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Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant.
(Microbiology Society, 2007) Owor, Betty E; Martin, Darren P; Shepherd, Dionne N; Edema, Richard; Monjane, Ade´rito L; Rybicki, Edward P; Thomson, Jennifer A; Vasani, Arvind
Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants – MSV subtypes A1–A6 – causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR–restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A1-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A1 recombination. One of the intra-MSV-A1 recombinants, designated MSV-A1UgIII, accounted for .60 % of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species.
Open Access
A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family
(BioMed Central, 2009) Varsani, Arvind; Shepherd, Dionne N; Dent, Kyle; Monjane, Aderito L; Rybicki, Edward P; Martin, Darren P
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
HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice
(BioMed Central Ltd, 2010) Pillay, Sirika; Shephard, Enid; Meyers, Ann; Williamson, Anna-Lise; Rybicki, Edward P
Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55Gag protein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1.
Open Access
Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice
(2014-05-24) Whitehead, Mark; Öhlschläger, Peter; Almajhdi, Fahad N; Alloza, Leonor; Marzábal, Pablo; Meyers, Ann E; Hitzeroth, Inga I; Rybicki, Edward P
Abstract Background Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. Methods In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. Results High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. Conclusions The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.
Open Access
Identification of two potyviruses of phaseolus vulgaris in South Africa
(1997) Van Tonder, Tertia; Rybicki, Edward P
A survey was conducted by researchers at ARC-PPRI on dry beans (Phaseolus vulgaris) during 1993. All the viruses known to occur on dry beans in South Africa were found, as well as a few unidentified viruses. Of these, samples 93/1 and 93/65 form the basis of this thesis. Electron microscopy (EM) indicated that these viruses could be potyviruses, as they were flexuous particles of approximately 700 to 800 nm. Observation of pinwheels in ultrathin sections of Nicotiana benthamiana infected with isolate 93/1 and Phaseolus vulgaris infected with isolate 93/65, confirmed that the viruses probably belonged to the Potyvirus genus, family Potyviridae. Further serological tests indicated that the viruses were related but not homologous to strains of clover yellow vein (CIYW) and blackeye cowpea mosaic (BICMV) viruses respectively. None of these viruses have previously been described as occurring in South Africa. As we were unable to positively identify the viruses with serological methods, we needed to characterise these viruses on a molecular level. Potyvirus specific oligonucleotide primers were used for PCR amplification of viral eDNA The primers amplify an approximately 700 bp fragment of the virus genome, spanning the 3' noncoding region as well as a part of the coat protein gene: one primer is complementary to the poly(A) tail, and the other to a sequence coding for a conserved block of amino acid sequences (also known as the WCIEN block) in the mid-region of the coat protein. The nucleic acid sequences of the PCR products were compared to that of other potyviruses to positively identify these isolates.
Open Access
Immunogenic assessment of plant-produced Human papillomavirus type 16 chimaeric L1:L2 virus-like particles and the production of an encapsidated therapeutic DNA vaccine candidate
(2017) Chabeda, Eva Aleyo; Hitzeroth, Inga I; Rybicki, Edward P
Cervical cancer caused by infection with Human papillomavirus (HPV) is the 4th most common cancer in women globally, and results in an estimated 266 000 deaths every year. Current vaccines are based on the immunodominant L1 major capsid protein, which assembles into virus-like particles (VLPs) that are highly effective in type-specific prevention of cervical infection. However, these vaccines are produced in expensive cell culture systems, are type-specific and do not induce the regression of established infections. The cervical cancer burden (~80%) is mainly in developing countries due to limited healthcare resources, therefore there is a need for more broadly protective and affordable vaccines. Plants provide an alternative platform to produce cheaper vaccines, given their scalability, rapid production and low risk of contamination. The L2 minor capsid protein has sequence regions that are highly conserved across several HPV types, and HPV-16 L2 peptides 108-120, 65-81, 56-81 and 17-36 have been shown to elicit cross-neutralising antibodies. To increase the immunogenicity of L2, second-generation L1:L2 chimaeric VLP (cVLP) vaccines have been investigated. In this study, the 4 L2 peptides above were used to generate plant-produced HPV-16-derived L1:L2 chimaeras. The L2 epitopes were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or between the helix 4 and β-J structural region at position 431 (SAE). All chimaeras were transiently expressed in Nicotiana benthamiana via Agrobacterium-mediated transfer. Optimisation of expression was conducted by comparing protein expression levels over several days using 4 plant expression vectors, with the highest yields obtained by targeting protein to the chloroplast or with the use of a self-replicating vector. The chloroplast targeted SAC chimaeras predominantly assembled into higher order structures (T=1 VLPs and T=7 VLPs), whereas SAE chimaeras assembled into capsomeres or formed aggregates, indicating that the length, sequence and substitution position of L2 epitopes affects VLP assembly. All SAC chimaeras in addition to SAE 65-81 (smaller epitope not previously tested in chimaeras) were used in vaccination studies in mice, and their immunogenic potential analysed in pseudovirion-based neutralisation assays (PBNAs). Of the 7 heterologous HPVs tested, cross-neutralisation was observed with HPV-11, -18 and -58. Only the anti-SAE 65-81 serum showed neutralisation of homologous HPV-16, suggesting that antibodies detected from all candidate vaccines were mostly non-neutralising, and that the position of the L2 epitope display is critical to maintaining L1-specific neutralising epitopes. Lastly, to address the lack of therapeutic efficacy of current vaccines, I aimed to develop a novel E7 DNA vaccine delivered by plant-made pseudovirions (PsVs). A geminivirus-derived self-replicating plasmid encoding a shuffled E7 (E7SH) sequence that has no transformation ability but contains natural cytotoxic T-lymphocyte epitopes, was constructed using Goldenbraid technology and co-expressed in plants with HPV-16 or HPV-35 L1- and L2-encoding expression vectors. The pseudogenome was successfully encapsidated into plant-made PsVs. These PsVs were capable of infecting mammalian cells and encapsidated replicons expressed E7SH showing the promise of this candidate vaccine as a future combination prophylactic and therapeutic vaccine.