Browsing by Author "Rybicki, Ed"
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- ItemOpen AccessAssessing TMV as an immunogenic particle : the expression of HIV-1 subtype C V3 loop neutralizing antibody epitopes on the surface of TMV virions(2005) Valley-Omar, Ziyaad; Rybicki, Ed; Jaffray, AnnIn an attempt to establish a plant-based Human immunodeficiency virus-1 (HIV-1) subtype C neutralizing antibody stimulating vaccine, a Tobacco mosaic virus (TMV) derived vector was used to express recognized HIV neutralizing antibody epitopes. These epitopes were expressed on the surface of the TMV coat protein, which served as an ideal means of antigen display. This model antigen display system was capable of assembling into multivalent, highly repetitive structures thereby displaying many copies of the attached epitope in the assembled virion. Three TMV-based vectors were acquired, which were essentially identical, differing only in the position at which they could accommodate a foreign protein fusion. These vectors allowed the display of a foreign peptide at the N-terminus, Cterminus or 60S-loop regions of the TMV coat protein. all of which protrude on the surface of the assembled virion. The HIV V3 loop is recognized as the principal neutralizing domain, and was the neutralizing epitopes displayed by the vectors. The epitope sequences used were derived from a cohort of infected individuals in Durban, South Africa, who displayed broad cross-neutralizing V3-specific activity towards heterologous viral strains. The recombinant viral vectors were shown to efficiently infect the host Nicotiana benthamiana plants and assemble into multivalent recombinant virion structures as observed by transmission electron microscopy. However, the level of coat protein expression was significantly dependent on the position of the coat protein fusion as either the levels of V3 epitope expressed or TMV coat protein was found to vary between the different vector types, confirmed by means of immunoblotting and enzyme-linked immunosorbent assays. Immunogenicity analysis using a guinea pig model was used to assess the ability of the recombinant vectors to firstly establish a V3-specific immune response, and secondly to stimulate a virus-specific neutralization antibody response. As a result of time constraints only the C-terminal coat protein fusions were assessed in the guinea pig model. Inoculated guinea pigs displayed distinct and gradually increasing V3-specific immune responses after 2 boosts. Serum samples that displayed the strongest V3 peptide responses were then analyzed for their ability to neutralize HIV infection in HIV pseudovirion neutralization assays. Results for selected serum samples showed no HIV neutralizing activity above what could be recognized as background activity. Thus the candidate vaccine, although establishing a path for the assembly of a multivalent vaccine, failed in its attempt to stimulate a neutralizing antibody response. This study has nevertheless paved a direct path to the development of variations of this type of vaccine possibly using different and perhaps more effective epitopes for candidate vaccine purposes.
- ItemOpen AccessBeak and feather disease virus candidate vaccine development(2012) Duvenage, Lucian; Rybicki, Ed; Hitzeroth, Inga; Meyers, Ann[Fix supervisors field.] Psittacine beak and feather disease, caused by a circovirus known as beak and feather disease virus (BFDV), is a threat to both wild and captive psittacine species. There is currently no vaccine against BFDV and safe and affordable vaccine candidates are needed to alleviate the disease burden caused by this virus. Production of the BFDV's major antigenic determinant, the capsid protein (CP), in the inexpensive and highly scalable plant expression system, could satisfy these requirements as a potential subunit vaccine. In this work, truncated CP (ÄN40 CP) was first expressed in E. coli to successfully generate anti-CP polyclonal antibodies. ÄN40 CP and full-length CP transient expression in tobacco (Nicotiana benthamiana) was optimised as fusions to elastin-like polypeptide (ELP). Fusion of CP or ÄN40 CP to ELPs of different lengths was shown to increase yield relative to unfused CP/ÄN40 CP. Free ELP and a GFP-ELP fusion could be purified by inverse transition cycling (ITC), using centrifugation and membrane filtration methods. A ÄN40 CP-ELP fusion expressed in plants could be partially purified and represents low-cost vaccine candidate against BFDV. A candidate DNA vaccine expressing ÄN40 CP was also evaluated for expression of the antigen in vitro and may prove useful in a prime-boost regimen together with one of the plant-produced vaccine candidates.
- ItemOpen AccessBioMed Central Membership(2012-05) Rybicki, EdBioMed Central is an internet publishing platform, recently purchased by Springer. They publish a suite of 233 Open Access peer-reviewed biomedical journals online, which are now joined by another, more social science-oriented set of offerings (81 journals) from SpringerOpen. BMC offers a subscription package – already taken up by the Universities of the Witwatersrand and Stellenbosch, and now also by UCT - which at the "top end" rate (above GBP20 000/yr), offers a 15% discount per article fee, and free repository connection to all institutional BMC-published articles, and a branded institutional web page linking to all the articles.
- ItemOpen AccessConstruction and characterization of chimaeric human immunodefiency virus type 1 subtype C Gag virus-like particles(2006) Halsey, Richard James; Tanzer, Fiona; Jaffray, Ann; Rybicki, EdIn this study I explored the possibility of making HIV-1 subtype C Pr55gag-based chimaeric virus-like particles (VLPs) as a boost to the HIV-IC multigene DNA vaccine pTHr.grttnC, which encodes a modified Gag-RT-Tat-Nef fusion protein (GRTTN). Furthermore, an attempt was made to produce VLP analogues to the HIV-IA polyepitope DNA vaccine pTHr.HIVA. A range of in-frame fusions with the C-termini of myristylation-competent p6-truncated Gag and native Pr55gag were made to test how the length of polypeptide and its sequence might affect VLP formation and structure.
- ItemOpen AccessDevelopment of candidate Human papillomavirus vaccines(2003) Varsani, Arvind; Rybicki, Ed; Williamson, Anne-LiseThe objective of this thesis was to investigate novel and plant-based vaccines against the Human papillomavirus type 16 (HPV-16), which is primarily responsible for cervical cancer. As a first study, the L1 gene of a South Africna variant of HPV-16 (L1 504) and a mutant (504[ΔA266T]), where the alanine at 266 was mutated to a threonine, were expressed in insect cells by recombinant baculovirus, and the resulting virus-like particles (VLPs) were tested with a panel of well-characterised monoclonal antibodies (Mabs).
- ItemOpen AccessDevelopment of plant-produced African horse sickness vaccines(2019) Dennis, Susan Jennifer; Rybicki, Ed; Hitzeroth, Inga; Meyers, AnnAfrican horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades, but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines, initially focusing on the use of insect and mammalian cell expression systems. More recently, several studies have demonstrated the potential for using plant expression systems for the production of virus-like particles (VLPs), which are excellent vaccine candidates, as they do not contain virus genetic material and are DIVA compliant. A vaccine alternative to the currently used live vaccine necessarily needs to provide protection against all nine serotypes of the virus. Cross-protection has been shown to exist between certain serotypes of the virus and as capsid protein VP2 is the protein responsible for AHSV serotype specificity, the idea of a plant-produced VLP vaccine containing a representative VP2 protein from each of the different serotype groups, was conceived. Such a vaccine would potentially provideprotection against all 9 serotypes of the virus and would have DIVA capability. Furthermore, it would address local concerns regarding the use of a live vaccine and would serve as a potentially acceptable prophylactic or rapid response antidote in the wider international context. This work describes two approaches in the development of VLP vaccines in plants. In the first part of this study, the ability of 2 different serotypes of plant-produced AHSV VLPs to safely stimulate an immune response in horses, was investigated. Co-infiltration of Nicotiana benthamiana plants with Agrobacterium constructs encoding the four AHSV serotype 5 structural proteins VP2, VP3, VP5 and VP7, was shown to result in assembly of complete VLPs. Furthermore, co-infiltration with the constructs, encoding VP3 and VP7, together with constructs encoding the two outer capsid proteins VP2 and VP5 of a second serotype, AHSV 4, resulted in assembly of complete AHSV 4 VLPs. Horses vaccinated with plant-produced AHSV 4 and 5 VLPs, all seroconverted after two doses of the vaccine and the virus neutralization titres indicated that the plant-produced VLP vaccines are likely to be at least as effective as the current LAV in protecting against AHSV 4 or AHSV 5. However, they have the added advantage of being free from any of the associated risks of a live vaccine, such as reversion to virulence or genetic re-assortment with field or vaccine strains. In the second part of the study, the use of the so-called SpyTag/SpyCatcher or bacterial “superglue” technology was investigated. This technology is based on the peptide SpyTag irreversibly coupling to the SpyCatcher protein, forming an isopeptide bond when the two are mixed together. The plant-based expression system was used to produce Spy VLPs consisting of either Acinetobacter phage (AP205) VLPs or tobacco mosaic virus (TMV) VLPs displaying a SpyTag or SpyCatcher peptide. In addition, AHSV 5 VP2 displaying SpyTag was expressed in plants and several coupling strategies were tested to determine whether AP205 particles displaying AHSV 5 VP2 could be formed as a result of binding between the SpyTag/SpyCatcher moieties of the recombinant proteins. Although it was not proven that coupling occurred, this research will pave the way towards developing a multivalent vaccine platform whereby VP2 of different AHSV serotypes can be displayed on the Spy VLP surface to allow optimal presentation of these proteins to the animal's immune system. Together, the results obtained in this study show that there is great potential for the production of novel, diverse, efficacious and economically viable AHSV VLP vaccines in plants.
- ItemOpen AccessDevelopment of West Nile virus candidate vaccines in Nicotiana benthamiana(2021) Wayland, Jennifer; Meyers, Ann; Chabeda, Aleyo; Rybicki, EdWest Nile virus (WNV) is a widely disseminated flavivirus, with a geographical range that now includes Africa, America, Europe, the Middle East, West Asia and Australia. The virus is vectored by Culex mosquitoes and is maintained in a bird-mosquito transmission cycle with hundreds of bird species acting as reservoir hosts. In humans, infections can develop into febrile illness and severe meningoencephalitis and to date, there is no treatment or vaccine available. In horses, approximately 20% of infections are symptomatic, of which 90% of cases involve neurological disease, with 30-40% fatality rates. Several veterinary vaccines specific to the lineage 1 WNV strains are commercially produced in America and Europe, however, these vaccines are not easily obtainable for low and middle-income countries (LMIC) due to their high cost and that associated with importation as well as the need for annual vaccination. Due to continuous global disease outbreaks in birds, humans and horse populations with no preventative measures for humans, WNV poses a major public health threat, especially in naïve populations. The development of a vaccine that contributes to the ‘One Health' Initiative could be the answer to prevent the spread of the virus and control the disease. Current veterinary vaccines are produced in expensive cell culture systems that require sterile conditions, high-level biosafety facilities and trained personnel for their preparation. Transient plant-based expression systems have proven to be a very cost-effective means of making complex proteins. Plants can produce and modify proteins in a similar manner to mammalian cells and production does not require sterile conditions or specialised facilities. We propose that plants could be a viable means of making feasible, low-cost reagents for WNV, specifically virus-like particles (VLPs) for use as vaccines in South Africa and other LMIC. In this study, we set out to develop two particulate candidate vaccines based on a virulent South African WNV strain using Nicotiana benthamiana as the expression platform. We aimed to develop the first candidate vaccine by exploiting the virus's ability to form noninfectious VLPs by expressing only the WNV membrane (prM – precursor, M – matured) and envelope (E) proteins. Infiltration of these recombinant plasmids into plants yielded no protein expression unless co-expressed with the human chaperone protein calnexin (CNX), upon which expression of both M and E proteins were observed. We investigated the assembly of prM and E into VLPs by transmission electron microscopy (TEM), however, purification of these particles proved difficult with poor reproducibility and VLP yield. This led to the development of an alternative candidate vaccine making use of the antigendisplay technology based on the SpyTag (ST) and SpyCatcher (SC) peptides. The immunodominant epitope of the WNV E protein, domain III (EdIII), was selected for antigen display. Two constructs of the EdIII gene were generated, one with the SC peptide on the 5'- (SC-EdIII) and the other on the 3' end (EdIII-SC). Both SC-EdIII and EdIII-SC proteins were successfully expressed in the presence of the human chaperone protein calreticulin, and purified with yields of 9 mg/kg and 69 mg/kg fresh leaf weight (FLW), respectively. The VLP core selected for the display of the SC-linked EdIII proteins comprised the coat protein of the bacteriophage AP205 with the ST peptide linked to its N-terminus (ST-AP205). Spytagged-VLPs were purified by density gradient ultracentrifugation at a yield of approximately 50 mg/kg FLW. The purified SC-linked EdIII proteins and ST-AP205 VLPs were coupled in vitro, but successful complex formation of AP205:EdIII was only observed between ST-AP205 and EdIII-SC and not when the SC peptide was located on the N-terminus of EdIII. We further demonstrated the successful complex formation of AP205:EdIII in vivo by coinfiltration of the EdIII-SC and ST-AP205 constructs, as well as by extracting leaves of plants infiltrated individually with either of the constructs. Due to the ease of purification and the high yields of AP205:EdIII achieved, the co-extraction process was optimised to obtain the best coupling yield possible by evaluating different FLW extraction ratios and the formation of VLPs was confirmed by TEM. The optimal co-extraction process was established at a FLW ratio of 1:2 ST-AP205 to EdIII-SC yielding approximately 23 mg/kg AP205:EdIII/FLW processed. In this study, we describe the successful production of two particulate candidate vaccines. The first is based on the expression of the WNV prM and E genes in the presence of human CNX and the second is based on the ST/SC antigen-display technology. These outcomes exhibit the potential plants have of being used as biofactories for making significant pharmaceutical products for the ‘One Health' Initiative and could be used to address the need for their local production in LMIC.
- ItemOpen AccessExperimental investigations of mastrevirus molecular biology and evolution(2008) Van der Walt, Eric; Rybicki, Ed; Martin, D PThis dissertation describes three major sets of experiments, all of which involved the construction and use of various reciprocal chimaeric MSV constructs. First, chimaeric viruses were used in genetic complementation-type experiments to investigate the biological significance of interactions between the two virion-sense open reading frames (ORFs) of MSV, their products, and the rest of the genome. Six chimaeric MSV constructs were made by reciprocally exchanging the ORFs encoding movement protein (MP) and coat protein (CP) individually, and in pairs, between MSV-Kom and MSV-Set, which share just 78% overall nucleotide identity. Analysis of symptomatology and infection efficiency of chimaeras and wild-type parental viruses revealed evidence of functionally relevant specific interactions between MSV MP and CP.
- ItemOpen AccessExpression of HIV-1 subtype C nef in E. coli and Nicotiana benthamiana : development of plant-based vaccines for HIV(2003) Bandawe, Gama; Rybicki, EdExpression of the nefgene from HIV-1 subtype C in Nicotiana benthamiana was carried out using a TMV -based vector with the aim of developing a plant-based candidate vaccine for HIV-1. The nef gene of the DU151 isolate of mV-l subtype C taken from a recently seroconverted individual was amplified by PCR with a deletion of 59 amino acids from the cytotoxic N-terminal. The amplified gene was inserted into a bacterial expression vector pProEXHTb for rapid expression of Nef protein, which was used as a diagnostic tool in the development of an indirect ELISA assay for detection of Nef in Nicotiana benthamiana. An indirect ELISA assay was developed using a commercially available polyclonal anti Nef antiserum raised in sheep. The role of codon optimization in expression of Nef in benthamiana was investigated. A synthetic nef gene was constructed based on the codon usage of benthamiana. The plant codon optimized gene and the wild type nef genes were inserted into the TMV -based vector pBSG1057. RNA transcripts from both constructs were used to infect young benthamiana plants. Expression of nefmRNA was confirmed by RT -PCR analysis of total RNA extracted from plants inoculated with respective constructs. The Nef protein was expressed at low levels which were detectable by ELISA. Nef was detectable by Western blot after concentration of plant extract using a membrane filter device. Quantitative analysis of Nef expression in plants was done by western blot on concentrated plant extract from three separate infections. Codon optimization of the nef gene improved the expression of Nef by a factor of about two.
- ItemOpen AccessExpression of HPV-16 L2 in plants(2008) Pereira, Roman; Rybicki, Ed; Becker, Inga; Maclean, JamesThis study demonstrates high level expression of L2, 25 mg.kg⁻¹of leaf material, is achievable. Interestingly, expression was best when coded for by a mammalian codon-optimized form of the L2 gene as opposed to the wildtype or plant codon-optimized (plantized) genes. Moreover, real time PCR revealed limited levels of transcript when coded for by the plantized gene in comparison to the other genes. A set of vectors which target the protein to the cytoplasm, the endoplasmic reticulum (ER), chloroplast or apoplastic space were Expression of HPV -16 L2 in Plants used and it was found that targeting of the protein had no effect on its expression levels.
- ItemOpen AccessExtinctions: Past and Present Week 1 - An abundance of bacteria(2017-03-17) Chinsamy-Turan, Anusuya; Rybicki, EdIn this video, Professor Anusaya Chinsamy-Turan interviews Professor Ed Rybicki, a microbiologist based in the Department of Molecular and Cell Biology at UCT. They discuss the history and diversity of microbes on earth and how they played a key role in the development of oxygen in the atmosphere. Professor Rybicki also outlines how essential microbes are for our survival, as they help us to digest food, produce vitamins, and fight off disease.
- ItemOpen AccessGeneration and characterization of HIV-1 subtype C candidate vaccines that will induce high titre antibody responses to HIV-1 envelope glycoprotein(2020) van Diepen, Michiel Theodoor; Williamson, Anna-Lise; Chapman, Ros; Rybicki, EdDespite huge strides being made towards decreasing the number of individuals getting newly infected with HIV-1, and in reducing AIDS-related deaths, unfortunately current predictions are that the 2020 UNAIDS goals (90-90-90 targets, where 90% of people living which HIV-1 are diagnosed as such, from which 90% will will receive sustained antiretroviral therapy, resulting in viral suppression in 90% of these individuals by 2020) are out of reach. This of course means that the numbers of newly infected indivuals and AIDS-related deaths will be above the target derived from the 2020 UNAIDS goals. The development of an effective HIV vaccine could therefore be an important step towards realising these objectives. In work done for this thesis, a heterologous HIV-1 vaccine platform regimen was developed using antigen sequences from the predominant circulating HIV-1 subtype (subtype C) in South Africa. Specifically, this involved use of the envelope glycoprotein sequence of the CAP256 superinfecting virus (CAP256_SU) from the CAPRISA 002 cohort, and a mosaic Gag sequence which resulted in robust autologous Tier 2 neutralisation of CAP256_SU pseudovirions. The envelope glycoprotein sequence was modified so as to replace the native leader sequence with the tissue plasminogen activator leader, the furin cleavage site with a glycine rich flexible linker, and to introduce an I559P mutation. DNA and modified vaccinia virus Ankara (MVA) vaccines were generated where Env was truncated to gp150, thereby retaining the transmembrane domain and a partial cytoplasmic tail (Env). The Env sequence for the protein vaccine was further trimmed by removal of the transmembrane domain to give gp140, leading to a soluble, secreted protein (soluble Env). This allowed for the latter vaccine to be affinity purified using lectin (soluble Env (GNL)), and after generating stable cell lines, soluble Env yields were high enough to enable size exclusion chromatography which allowed isolation of the trimeric fraction of Env as determined by molecular weight (soluble trimeric Env). A Cterminal His-tagged version of soluble Env was generated as well. Surprisingly, the folding of Env-His was inferior to soluble Env, with a switch in profile from mainly trimeric Env to mainly monomeric Env. Nevertheless, soluble Env-His (GNL) and soluble trimeric Env-His were assessed for the presence of Env broadly neutralising antibody (bnAb) epitopes in an ELISA assay. The V3-glycan supersite (binding of bnAbs PGT128 and PGT135), the CD4-binding site (VRC01) and the V2-glycan site (PG9) were detected for both Env-His (GNL) and soluble trimeric proteins, whereas low signals for PG16, PGT145 and CAP256-VRC26.08, bnAbs which specifically recognise Env trimers in a native-like conformation, were only detectable for soluble trimeric Env-His. Soluble Env (GNL) was subsequently used as a protein vaccine in rabbits to test the immunomodulatory effects of the two adjuvants AlhydroGel (similar to alum) or the MF59-like squalene-based oil-in-water nano-emulsion AddaVax. Soluble Env (GNL) adjuvanted in AlhydroGel resulted in improved immune response in rabbits, with significantly higher serum binding antibodies to soluble Env (GNL) and scaffolded CAP256 V1V2-loop in comparison to AddaVax and unadjuvanted protein. Furthermore, significantly higher neutralisation titres to Tier 1A subtype C virus (MW965.26), in combination with an improved breadth to subtype C Tier 1A and 1B viruses, were observed in the AlhydroGel group. However, no neutralisation of Tier 2 viruses was detected. Nonetheless, AlhydroGel was selected as the best protein adjuvant for all further rabbit immunogenicity studies. Furthermore, in all subsequent experiments, soluble trimeric Env was used as a protein vaccine. DNA and recombinant MVA vaccines were generated using a membrane anchored gp150 (Env) with the aim that co-expression with mosaic Gag (GagM) would lead to the incorporation of Env into Gag virus-like particles (VLPs). Electron microscopy of cells expressing Env+GagM from DNA and recombinant MVA vaccines verified VLP formation from these constructs, and the presence of Env was observed in VLPs purified using a two-step OptiPrep gradient centrifugation protocol. The presence of Env bnAb epitopes in cellular membrane-bound Env was verified by qualitative immunofluorescent microscopy of live-cell stainings and a quantitative FACS assay. The same bnAb epitopes as for the Env protein vaccine were detectable, including bnAbs recognising only native-like Env trimers (PG16, PGT145 and CAP256-VRC26_08). However, expression levels of native-like Env trimers were lower, at approximately 20% when normalised to VRC01. These HIV-1 DNA, rMVA and soluble trimeric Env protein vaccines were tested in different heterologous vaccine platform immunogenicity studies in rabbits. These consisted of either priming with two recombinant MVA vaccines and boosting with three protein vaccines (MMPPP), or priming with DNA vaccines followed by two MVA vaccines, followed by two protein vaccines (DDMMPP). Furthermore, the inclusion of GagM into the DNA and MVA vaccines was compared to use of Env alone. Both vaccine regimens resulted in binding antibodies to soluble trimeric Env and a scaffolded CAP256 V1V2-loop; however, these were induced by MVA and protein vaccines, but not by DNA vaccines. Despite the lack of Env binding antibodies after DNA vaccination, better neutralisation was observed for the DDMMPP regimen compared to MMPPP, resulting in higher sera neutralisation titres towards vaccinematched, autologous Tier 2 CAP256_SU virus. Most encouragingly, when compared to Env alone, the inclusion of Gag (Env+GagM) into DNA and MVA vaccines improved the immunogenicity of the DDMMPP regimen even further. For Env+GagM DDMMPP, more animals developed Tier 2 neutralising antibodies, and improved titres, whereas Tier 2 neutralisation in general started to develop after fewer vaccinations, as for most rabbits this was observed after the second MVA inoculation. In an attempt to improve the spike density of Env on VLPs and the plasma membrane, two Env chimaeras were made replacing parts of gp41 with the corresponding elements of influenza A H5 haemagglutinin (HA2) (Env:HA2 chimaeras). Increased Env spike density was observed in a previous study using this strategy for the gp41 transmembrane domain and cytoplasmic tail (gp140HA2tr). A similar construct was generated here for CAP256_SU and a second chimaera was included replacing the whole of gp41 with HA2 (gp120HA2). Surprisingly, in experiments where VLPs were purified from OptiPrep gradients or the whole-cell bnAb FACS assay conducted with these Env:HA2 chimaeras, there was no evidence of increased spike density on VLPs or the plasma membrane as compared to Env. Furthermore, the folding of Env was severely impacted, especially regarding gp120HA2 where no binding of PG16, PGT145 and CAP256 VRC26.08 - bnAbs recognising native-like Env trimers - was observed. Although results for gp140HA2tr was improved over gp120HA2, in general the data for gp150 (Env) was superior in both the bnAb live-cell staining and FACS assay. Consequently, when both Env:HA2 chimaeras in combination with GagM were tested in the DDMMPP regimen, no improvement was observed with regard to autologous Tier 2 neutralisation. For rabbits receiving gp120HA2, no animals developed Tier 2 nAbs, whereas for gp140HA2tr, Tier 2 neutralisation in general developed later and to lower titres compared to Env+GagM. In conclusion, different HIV-1 DNA, recombinant MVA and protein vaccines were generated and characterised both in vitro and in vivo, leading to a vaccination regimen that induced both high titre Env binding and vaccine-matched Tier 2 neutralising antibodies in rabbits. Furthermore, a new Env sequence, the first from the South African CAPRISA cohort, has been added to the small list of Env sequences that can induce Tier 2 neutralisation.
- ItemOpen AccessHPV pseudovirion production in plants(2013) Kennedy, Paul; Rybicki, Ed; Hitzeroth, Inga; Meyers, AnnHuman papilloma virus (HPV) infection is the most common etiological agent of cervical cancer, the most common cancer in women in Africa. The lifecycle of HPV has historically made the virus difficult to culture in vitro, and this has hindered the study of the virus, as well as development of vaccines. The development of synthetic HPV particles, such as virus-like particles (VLPs) and more recently pseudovirions (PsVs), has allowed for unprecedented insights into the lifecycle and immunology of this virus. This has led to the development of two currently available vaccines, namely Cervarix™ and Gardasil®. Cervarix offers protection against high-risk HPV types 16 and 18, while Gardasil offers further protection against types 6 and 11. Both of these vaccines are based on major capsid protein L1 Virus-like particles (VLPs). While these vaccines show no loss of efficacy, further work is underway to develop a second generation HPV vaccine that is cheap, stable and displays cross-neutralising activity across a broader range of HPV types. The recent efficient methods for intracellular production of HPV PsVs encapsidating nonpapillomaviral DNA (pseudogenomes) has allowed for development of a robust and sensitive pseudovirion-based neutralisation assay (PBNA), which has become the gold standard neutralisation assay for the testing of candidate HPV vaccines. The currently accepted PsV production method utilises mammalian cell culture to produce HPV PsVs, encapsidating a SEAP reporter plasmid, at high titres. While this is an effective method of PsV production, mammalian cell culture is expensive and time-consuming. Transient recombinant protein expression in plants offers a rapid and cost-effective alternative to mammalian cell culture. Here, we developed a method of high-titre HPV PsV production in plants. The autonomously replicating plant vector, pRIC3, was modified to include mammalian reporter cassettes encoding luc or SEAP, for the production of reporter pseudogenomes DNA in plants by Agrobacterium-mediated transient expression. The SEAP and luc cassettes were introduced into pRIC3 upstream of the plant cassette, which was included only to increase the final pseudogenome size for efficient packaging into PsVs. The SEAP cassette was also introduced into pRIC3 in place of the plant cassette, to form a smaller pseudogenome. Thus three vectors were created, namely pRIC3-mSEAP+ (6.4Kbp pseudogenome), pRIC3-mluc+ (7.4Kbp pseudogenome), and pRIC3-mSEAP (4.8Kbp pseudogenome), which would produce pseudogenomes that covered the full range of plasmid sizes incorporated by assembling HPV capsid proteins in vivo. All three replicating vectors demonstrated the formation of a replicon, and autonomous replication, in Nicotiana benthamiana plants. Each of these vectors were co-infiltrated with the non-replicating transient plant expression constructs pTRAc-hL1 and pTRAc-hL2, which encode human-codon optimised forms of HPV-16 major and minor capsid proteins, respectively. It was expected that encapsidation of replicon DNA as a pseudogenome into assembling HPV particles would result in the production of HPV PsVs in planta. In addition, L1 and L2 were expressed in the absence of replicon DNA to form L1/L2 VLPs. Particles were extracted from plant material at four days post-infiltration, using a modified VLP extraction protocol. HPV particles were separated on the basis of isopycnic caesium chloride density gradient ultracentrifugation, dialysed against high-salt PBS and identified by fractionation and probing with an anti-L1 antibody. Particles corresponding to the buoyant density of pseudovirions were seen in samples with or without replicon DNA. Western blotting showed that all particles had incorporated both L1 and L2 proteins. Particles were digested with proteinase K to release encapsidated pseudogenome DNA and PCR confirmed the presence of replicon-specific DNA in each PsV. Electron microscopy confirmed the presence of HPV-16 PsVs in all samples. To test whether plant-produced HPV-16 PsVs could be used in pseudovirion-based neutralisation assays, mammalian cells were pseudoinfected with purified mSEAP, mSEAP+ or mluc+ PsVs. mSEAP and mluc+ PsVs elicited a reporter gene response in mammalian cells 72 hours post-infection using SEAP and luciferase assays, respectively, while mSEAP+ PsVs showed no reporter gene expression in mammalian cells. PsVs incubated with a known HPV-16 neutralising antibody showed partial neutralisation of mSEAP PsVs and complete neutralisation of mluc+ PsVs To our knowledge, this is the first demonstration of production of HPV PsVs in plants, and their use in a PBNA. Further, it is the first demonstration of production of HPV L1/L2 VLPs in plants. While much work remains to improve plant production and purification methods of PsVs, as well as mammalian expression following PsV pseudoinfection, this is an important step towards a new method of PsV production.
- ItemOpen AccessHuman papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa: a cross-sectional study(BioMed Central Ltd, 2009) Moodley, Jennifer; Constant, Deborah; Hoffman, Margaret; Salimo, Anna; Allan, Bruce; Rybicki, Ed; Hitzeroth, Inga; Williamson, Anna LiseBACKGROUND:Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. METHODS: A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. RESULTS: The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 - 57.7) for those that were HC2 positive and had a viral load of [less than or equal to] 181.1 RLU (the median HPV viral load), and 33.8 (95% CI 6.4 - 178.9) for those that were HC2 positive with a HPV viral load > 181.1 RLU. CONCLUSION: Women initiating ARVs have a high prevalence of abnormal Pap smears and HR-HPV. Our results underscore the need for locally relevant, rigorous screening protocols for the increasing numbers of women accessing ARV therapy so that the benefits of ARVs are not partially offset by an excess risk in cervical cancer.
- ItemOpen AccessAn in depth study of human papillomavirus diversity in South African women infected with HIV(2009) Salimo, Anna T; Hitzeroth, Inga; Williamson, Anna-Lise; Rybicki, EdCervical cancer is the second most common cancer affecting women and in most developing countries it remains the leading cause of cancer deaths. In South Africa, more than 3 400 women succumb to the disease every year and 1 in 31 women develop cervical cancer. The causative agent for cervical cancer is the Human papillomavirus (HPV). High-risk (carcinogenic) HPV types have been linked with 99% of the incidences of cervical cancer. The most common types identified in almost 70% of cervical cancer cases worldwide are HPV 16 and 18. HPV infection is very common in young healthy women and most immunocompetent individuals can clear HPV infection. However, in immunosuppresed women, clearance by host immune system is impaired. In addition, multiple HPV infections are quite common in women with Human immunodeficiency virus (HIV) infections. The objectives of this study were to identify HPV types in South African women who also had HIV infection, and secondarily, to determine if recombination of HPV genomes occurs. Determining the HPV types circulating in this country is important to enable identification of most common HPV types, in order to guide the development of vaccines against HPV infection. HPV genotyping was performed by the commercial Roche Linear Array HPV Genotyping Test.
- ItemOpen AccessIntroduction to Molecular Virology(2014-09-16) Rybicki, EdIntroductory Virology for 2nd and 3rd year courses. The material consists of a series of linked pages exploring an introduction to the concept of viruses, and an exploration of their general properties. Virology teaching material used for University of Cape Town Molecular and Cell Biology Department courses. This site provides the basis of material for 7-lecture course in introductory microbiology (MCB2016F) and a 20-lecture course (MCB3024S, Defence and Disease) given to third-year students.
- ItemOpen AccessAn investigation into improved HIV-1 subtype C envelope based vaccine design(2014) Margolin, Emmanuel Aubrey; Williamson, Anna-Lise; Rybicki, Ed; Chapman, Ros; Meyers, AnnIncludes abstract. Includes bibliographical references.
- ItemOpen AccessAn investigation into the development of plant-derived vaccines against human papillomavirus type 11 and Cottontail rabbit papillomavirus(2004) Kohl, Thomas Oliver; Rybicki, Ed; Becker-Hitzeroth, Inga IThe principal object of this thesis was to investigate the feasibility of developing a novel plant-derived vaccine against Human papillomavirus type 11 (HPV -11), the causal agent of genital warts and laryngeal condylomas. A secondary objective was a proof-of-concept study using Cottontail rabbit papillomavirus (CRPV), as this is a viable animal model for human papillomavirus vaccines. Accordingly, I investigated and compared the potential of two different plant expression systems.
- ItemOpen AccessInvestigation of particulate HIV-1 Env vaccine candidates using Zera® and SpyTag/SpyCatcher technologies(2022) Ximba, Phindile Thobeka; Rybicki, Ed; Williamson, Anna-Lise; Meyers, AnnThe HIV-1 envelope glycoprotein (Env) is the primary focus of prophylactic HIV vaccine development. However, the unusually low density of Env spikes on the virion (≈14 spikes/virion) is unfavourable for eliciting high titre, long-lasting antibody responses. It is possible that increasing the Env spike density of particulate vaccine candidates generated by protein body formation or via the display of Env on nanoparticles could improve the induction of long-lasting neutralising antibodies (NAbs). For this thesis, two different nanoparticle approaches were therefore investigated. The HIV-1 Env sequence used for both approaches was derived from the superinfecting subtype C CAP256 virus. This was truncated to remove the transmembrane domain, and engineered to contain a flexible linker (FL) in place of the furin cleavage site and an I559P mutation to generate soluble, stable and cleavageindependent gp140 proteins. The first approach investigated the impact of genetically fusing a 27 kDa proline-cysteine-rich domain of the ɣ-zein maize seed storage protein - Zera® - to either the N- or C-terminus of CAP256 gp140. Fusion of Zera® to a protein of interest can promote the self-assembly of large protein bodies (PBs) containing the protein of interest, thereby improving yields of the recombinant protein and enabling easy isolation using gradient ultracentrifugation. The purification of Zera-induced Env PBs from infiltrated Nicotiana benthamiana plants was not optimal. Consequently, the generation of Zera®-induced gp140 protein bodies was evaluated in a mammalian expression system. Stable HEK293 cell lines expressing Zera®-gp140 or gp140-Zera® were generated. A mixture of small PB-like structures was observed in cells expressing gp140-Zera®. However, no PB-like structures were seen in cells expressing Zera®-gp140. The immunogenicity of Zera®-gp140 and gp140-Zera® was evaluated by in rabbits. Binding and Tier 1A neutralising serum titres were higher for gp140-Zera® than for Zera®-gp140. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralised a Tier 1B pseudovirus or the autologous Tier 2 CAP256SU pseudovirus, suggesting that Zera® might have compromised the structure of the Zera®-tagged gp140 proteins. The second approach investigated the two-component SpyCatcher/SpyTag technology. The stable HEK293 cell line expressing CAP256 gp140-SpyTag (gp140-ST) was generated, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in E. coli and purified by ultracentrifugation. The gp140-ST trimers and the SCAP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). SDS-PAGE, dynamic light scattering and negative stain electron microscopy indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs. The second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens and then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. These results demonstrate that careful selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of HIV-1 envelope trimers is an important consideration and that this could improve the effect of nanoparticle-displayed gp140.
- ItemOpen AccessInvestigations of the molecular determinants of maize streak virus replication(1999) Willment, Janet Anne; Rybicki, EdGeminiviruses replicate via a rolling circle mechanism, which initiates at the origin of replication located within the long intergenic region (LIR). The viral replication associated-protein (Rep) in conjunction with the host's DNA replication machinery is responsible for the initiation and termination of the replication cycle from a stem-loop structure, located within the LIR and conserved throughout the three genera of Geminiviridae. The specific interaction between the Rep protein with sequences within the intergenic region has been well characterised for the begomoviruses and to some extent the curtoviruses; however, this interaction in the mastreviruses, and in particular maize streak virus (MSV), has yet to be fully explored. A theoretical model has been proposed based on sequence data and informed by the current understanding of replication specificity in begomoviruses. Due to the lack of conservation of the stem sequence of the stem-loop structure amongst mastreviruses, the model implicates a pair of nucleotide sequence repeats called iterons. These are located within the stem structure, and on the complementary sense side of the LIR. The former is the putative site of Rep interaction with the LIR. These iterons would therefore potentially act as the determinants of replication specificity amongst mastreviruses.