PORTRAIT Human Vaccines & Immunotherapeutics 11:11, 2517--2521; November 2015; © 2015 Taylor & Francis Group, LLC
From plant virology to vaccinology: The road less travelled Edward Rybicki* Biopharming Research Unit; Department of Molecular & Cell Biology and Institute of Infectious Disease and Molecular Medicine; University of Cape Town; Cape Town, South Africa
*Correspondence to: Edward Rybicki; Email: ed. [email protected] http://dx.doi.org/10.1080/21645515.2015.1092751 www.tandfonline.com
The beginning of my interest in science was when I became fascinated with chemistry at an early age, by helping test the pH of my primary school’s swimming pool: as a child in the Zambia of the mid-1960s, this was quite an important consideration, as I spent a large part of my leisure time in it! I went on to develop a keen interest in medical and other sciences, by way of voracious reading: our little library at home was soon exhausted, but the Lusaka Public Library held me for a while. An early exposure to science fiction by way of a family friend also started a passion that has not yet waned – and further exposure to science, by way of Isaac Asimov, Poul Anderson, Robert Heinlein, Arthur C Clarke and other “hard science” exponents of SF. Senior school in England, Zambia and finally Zimbabwe cemented my desire to be a scientist, with an exposure to mathematics, physics, chemistry and biology at A-level – but it was biology that eventually won, due to the fact it was simply so much more interesting as a fact-rich environment, and biochemistry in particular. I took that interest to the University of Cape Town in 1974, where I quickly learned that I couldn’t do medicine or biology as I had registered too late, but that I could do botany. After an excruciatingly boring year during which I repeated much of my last year of school, doing maths, physics and chemistry, and managed to avoid learning too much about floral plant diversity or dissection, I discovered that now I could do medicine if I wanted to. I declined – I still hope this wasn’t a mistake – as biochemistry and chemistry were now beckoning, with microbiology as filler. Chemistry taught me an appreciation for stereochemistry, mechanism and molecular structure; biochemistry cemented the structure and molecule-as-mechanism Human Vaccines & Immunotherapeutics
basics – but surprisingly it was microbiology that really appealed to me, with glimpses of the different kinds of biology that could result from integrating the same sorts of molecules. This derailed my career plans somewhat, as I was locked into majoring in chemistry – and it now looked like I was going to have to do three majors, if I was to continue with microbiology. It ended up being no contest: given bacteriology with a whiff of immunology and virology in my first semester, I happily tossed biochemistry – and I have never looked back. I was also fortunate in having some inspirational teachers, one of whom – Marc van Regenmortel – I am still in professional contact with.
Plant Virology phase (1977 – 1987) I specialised in Virology with a plant virus serology project in my Honours degree in 1977 – a separate 1-year degree in South Africa, during which I tossed biochemistry again. I did another plant virology and serology project for a Masters degree from 1978 – 1979, during which I was introduced to advanced physical analytics of the bromoviruses, including by means of analytical ultracentrifugation and electrophoresis, and also to the then very novel ELISA techniques – techniques that still serve me well today. I then branched out into a more general molecular plant virology project for a PhD (1980 – 1984), in which I explored the purification and characterisation of vanishingly small amounts of barley yellow dwarf and other viruses from wheat and barley plants, and the use of western blotting as 2517
About Edward Rybicki Dr. Rybicki received his pre- and postgraduate education from the University of Cape Town, South Africa, where he has worked throughout his career, gaining experience as a visiting scientist at Plant Genetic Systems, Ghent, Belgium (1984), Boyce Thompson Institute, New York, U.S. (1990–1) and Arizona Biodesign Institute, U.S. (2010). He became a full Professor in Microbiology in 2003, was a founder member of the Institute of Infectious Disease and Molecular Medicine in the Faculty of Health Sciences, and is currently Director of the Biopharming Research Unit in the Molecular & Cell Biology Department at the University of Cape Town. His research originally focused on molecular characterization of plant viruses, which brought about pioneering discoveries in recombinant gene expression in plants and in insect cells. Dr. Rybicki developed these “biopharming” techniques to produce candidate vaccines against HPV-16, HIV-1 subtype C, influenza H5N1, and other viruses. He has authored >120 papers and 47 patents. He is a recipient of numerous awards and honors, including the President’s Award of the South African Foundation for Research Development (1985) and the Meiring Naude Medal of the Royal Society of South Africa (1986).
an analytical tool – also skills that were to serve me very well later – as well as accidentally co-discovering a novel aphid virus. I stayed at the University of Cape Town (UCT) despite the chance of going to the US for a PhD, because I got married to a UCT postgraduate student and got appointed to a Lectureship in Virology at UCT in 1981, before I had finished my PhD. I ended up effectively doing a postdoc in my own lab, as the award of my PhD coincided with my first major research grant, the so-called President’s Award from the local Foundation for Research Development. This allowed me to retool my expertise with a short sabbatical in a biotech company in Ghent, Belgium (Plant Genetic Systems) in order to learn the ins and outs of DNA cloning. I continued with my serological interests, however, including helping apply ELISA to Streptomyces taxonomy, applying western blot techniques to the affinity purification of monospecific antibodies, and discovering pathogenesis-related PR-1-type proteins in maize and other plants by western blotting.
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Molecular Plant Virology and PCR phase (1988 – 1996) In 1988, and on the strength of my newly acquired DNA manipulation skills, I staked a bold but in retrospect rather na€ıve claim on a plant virus – in the complete absence of any published work from our group on it. I wrote a review1 on Maize streak virus (MSV): this is a small single-stranded DNA plant virus that was discovered and largely described from Africa over some 70 years, and I was, in my mind at least, claiming it back from foreign domination. In fact, it would take my group and my colleague Ralph Kirby only another year to characterize three novel strains of the virus, and to apply restriction map-based phylogenetic analysis techniques to determining their evolutionary relationships with other MSVs. At the same time, I was collaborating with my ex-PhD supervisor Barbara von Wechmar and our PhD students on molecular characterisation of aphid picorna-like viruses, and plant potyviruses. In 1990 I did a year-long sabbatical at the Boyce Thompson Institute for Plant
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Research in Ithaca, NY, with Steve Howell – and significantly expanded both my professional network and my skills base, with a nucleic acid-intensive set of projects largely based on the then very new polymerase chain reaction (PCR) technique that I had just started using. My geminivirus work evolved as my group and its molecular expertise grew, with characterisations of new potyviruses and geminiviruses by cloning and sequencing, and applications of PCR to detection and characterisation of diverse potyviruses and cereal-infecting geminiviruses – and also human papillomaviruses, with my recently-acquired medical virologist wife Anna-Lise Williamson.2-4 This work led to my developing a sideline in studying virus evolution, with major phylogenetic analyses published on both potyviruses and geminiviruses – and some collaboration on HIV-1 variation, with my former PhD student Carolyn Williamson.5 In a development that proved highly important for later work, I added a plant biotechnology string to my bow in this period, with supervision of projects involving making virus-resistant transgenic plants.
The Plant Biotechnology phase (1997 – 2002) While I continued to work with an expanding group on the molecular biology of geminiviruses in this period, and on transgenic resistance to viruses in plants,6 I had also begun with my student Kenneth Palmer to look at using geminiviruses for “molecular farming”, or the production of high-value biologics in plants.7 Work on MSV as an expression vector in cultured maize cells was especially useful,8,9 as it laid important groundwork for later molecular farming developments with another geminivirus. In particular, we were able to show stable, long-term (3 years) high-level expression of foreign genes in plant cells from plasmid-like circular dsDNA replicating forms of engineered MSV genomes. I was also prompted by Anna-Lise to “. . .express something useful!” when looking for a candidate gene to express in tobacco
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plants for molecular farming purposes – and we chose the HPV-16 L1 major capsid protein, both for reasons of familiarity due to our PCR work, and because we had the idealistic notion that we could make a cheap vaccine against cervical cancer in competition with Merck and GSK. During this time my lab also established a capacity to produce recombinant proteins in insect cells via engineered baculoviruses – ironically, as a means of providing “gold standard” geminivirus non-structural proteins for looking at MSV molecular biology.
The Early Vaccinology phase (2000 – 2009) Anna-Lise and I had received major local funding in 2000 to work on two completely different sets of vaccines: one grant, from the newly-formed South African AIDS Vaccine Initiative (SAAVI), was for the development of novel vaccines for South African variants of HIV-1 subtype C; the other, from the National Research Foundation (NRF)’s Innovation Fund, was for novel HPV vaccines. This stemmed from an idealistic desire on our behalf to try to make novel and affordable vaccines for Africa, given the complete absence of any manufacturing capacity in South Africa, as we had a spectrum of platforms for production of candidate vaccines between us – and given the opportunity to apply for two grants, we did, in the belief that we might get one. We were very pleasantly surprised, therefore, to get both! The SAAVI grant was renewed twice, to end in 2009, and the first NRF Innovation Award was followed by another specifically on plant- and insectmade HPV vaccines, and then other awards from the Poliomyelitis Research Foundation (PRF, South Africa), the SA Medical Research Council and the Cancer Association of SA (CANSA). My part of both projects had both insect cell and plant production arms: with HIV our object was to make budded virus-like particles (VLPs) from expression of the HIV-1 subtype C Pr55Gag polyprotein, as a booster protein vaccine for priming vaccinations with a DNA- or recombinant poxvirus- or BCG-vectored
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polygene encoding a Gag-RT-Tat-Nef fusion protein. With HPV we wanted to make generic L1 VLPs in plants as a cheaper and potentially orallyadministrable vaccine, novel chimaeric L1:L2 fusion proteins in insect cells as a second-generation vaccine with less type specificity, and to express L1 protein in BCG as another approach to making a cheap vaccine. My HPV work was quite successful from the outset: for plant expression, my team – co-supervised most ably by Inga Hitzeroth – was among the first in the world to make HPV-16 L1 VLPs in transgenic plants10; the first to show proof of efficacy in a rabbit model for a plantproduced L1 vaccine – for cottontail rabbit papillomavirus, CRPV11 – and the first to use a rTMV-vectored transient expression approach for any papillomavirus L1 (CRPV and HPV).11,12 Arvind Varsani in my group also successfully investigated, via baculovirus expression in insect cells, various surface locations in the L1 protein of HPV-16 for their potential to display a L2 peptide (108–120, LVEETSFIDAGAP) known to elicit widely crossneutralising antibodies.13 James Maclean went on to show that Agrobacteriummediated transient expression of HPV-16 L1 in Nicotiana benthamiana could be very significantly enhanced by use of human codon optimisation and localization of the protein in chloroplasts.14 HIV Gag expression work in insect cells was also highly successful: we were possibly the first to make HIV-1 subtype C Pr55Gag VLPs via baculovirus expression in insect cells, and showed that use of a matched gag DNA vaccine / Pr55Gag prime/boost regime gave excellent cytotoxic T-lymphocyte responses to Gag.15 We also showed that it was possible to express as VLPs the whole HIV Gag-RTTat-Nef (Grttn) polyprotein that was the basis of the SAAVI DNA/MVA vaccines being developed at the same time16 – but that GagRT or Gag-Tat-Nef (GagTN) had better yields and gave more uniform particles. We developed Gag VLP production into a reliable and reproducible process, with considerable optimisation of cell culture and other parameters17,18 – which led to our supplying our partner lab with VLPs for a series of landmark prime/
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boost experiments, involving a gag DNA/ Gag VLP combination19 and a gag BCG/ Gag VLP combination in two baboon experiments,19,20 showing that the combination was highly immunogenic, and induced a broad and polyfunctional central memory T-cell response appropriate for the control of HIV infection. Plant production of HIV-1 Gagderived antigens was less successful: yields of whole Pr55Gag were very low despite all optimisation efforts, and the best Ann Meyers in my group could do was prove production of VLPs – in itself a world first21 – and show that a MA/CA (p17: p24) fusion protein had some potential as a plant-produced HIV antigen.22 Something that came out of this work as a sideline, but which is proving to be highly useful in our continuing studies, was the development by Fiona Tanzer of an enhanced expression/DNA vaccine vector: this was based on a short enhancer element derived from the ssDNA Porcine circovirus 1, which increases protein expression in transfected cell cultures at least tenfold, and allows similar reductions in dosage of DNA vaccine required to elicit Grttn responses in mice.23 This would potentially allow significant dosesparing with the DNA vaccines involved in the SAAVI vaccine project,24,25 which went to clinical trial in 2008. Another sideshow project that has ended up being highly productive was the exploration of the potential of plant expression to produce pandemic influenza vaccines. As a result of a conference held in Cape Town in 2005, where a WHO influenza expert warned us “When the pandemic comes, you in the developing countries will be on your own”, we applied for extraordinary funding from the PRF in SA to explore the possibility of making a pandemic flu virus vaccine in South Africa. We chose the highly pathogenic avian influenza virus A H5N1 type haemagglutinin (H5 HA) as a target, and James Maclean was again instrumental in designing and successful early testing of plant-made soluble and membrane-bound forms. Further funding from the PRF and the SA MRC allowed proof of principle that we could in fact produce flu virus vaccine candidates in South Africa – both as subunit protein26 and as DNA vaccines.27
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In retrospect, while these projects were impossibly ambitious and not a little na€ıve, we and our co-workers received a crash course in both research vaccinology and the handling of big projects that has been crucial for all our subsequent work. We were also able to establish stable and wellqualified teams of people, with a nucleus of senior scientists who have been around us for up to 15 years. Another very important lesson was that we should patent our discoveries: in my case, this has led to me and my co-workers having the largest patent portfolio at our institution, and the largest molecular biotechnology-related portfolio in Africa – most of them to do with vaccines (14C patent families). The development of a set of well-tried protocols around expression of novel antigens in a variety of systems21 has also been invaluable – especially when funding circumstances demanded that we change direction.
Molecular Farming (2009 – 2015) Attendance of the very stimulating biennial Plant-Based Vaccines and Antibodies (now Biologics as well) Conferences since 2005 has led to very productive networking opportunities for us – as well as excellent funding opportunities. My group was fortunate enough to be included in a successful EU Framework 7 project called PlaProVa – Plant-Produced Vaccines (http://www.plaprova.eu/)-led by George Lomonossoff from the John Innes Centre in the UK, which involved us working on the development of plantmade vaccines against the newly-emerging Bluetongue virus (BTV) of sheep in Europe,28 as well as on plant production of our already-established HPV L1:L2 chimaeric vaccine candidates.29 We furthered ideas gained from the PBVAB on therapeutic vaccines for established HPV infections and cervical cancer into a collaborative project with Era Biotech in Spain, which resulted in a patent on the use of a maize zein seed storage proteinderived peptide (Zera) that causes protein body formation to be used as an adjuvant, for a shuffled HPV E7-derived synthetic protein vaccine.30 A happy marriage of my old plant virus interests with molecular farming occurred
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with the use of a mild strain of a beaninfecting geminivirus we had previously characterised as a replicating expression vector for tobacco, delivered by Agrobacterium31: our previous experience with MSV allowed rapid development of a versatile and high-yielding vector system that has been a highly valuable addition to our armamentarium. These projects have led on to more opportunities: the Technology Innovation Agency (TIA) and government Department of Science and Technology (DST) in South Africa have just funded us to work on novel BTV vaccines as well as vaccines for its fellow emerging orbivirus African horsesickness virus (AHSV), both major causes for concern in southern Europe as global warming progresses. Medicago Inc – possibly the world’s leading molecular farming vaccine company right now – is funding us to work on next-generation plant-produced HPV vaccines, and has patented a plant-made human rotavirus partially based on work done by us. We receive SA MRC and PRF funding for “One Health”-type projects on CrimeanCongo haemorrhagic (CCHFV) and Rift Valley fever (RVFV) bunyaviruses, where we are exploring the use of plants to make reagents as well as vaccines for these disease agents. We continue, on sniffs of funding from all over including patent royalties, to try to make vaccines against Beak and feather disease circovirus (BFDV), a ssDNA pathogen of parrots that threatens an endangered local species.32,33 Finally, we have branched out into reagent-based molecular farming, with the transient production in tobacco of recombinant horseradish peroxidase as a speciality reagent.34
The Future The potential importance of molecular farming for human health has been underlined recently with the apparently successful use of plant-produced MAbs (ZMapp) against Ebola virus disease in West Africa, and the proof of large-scale and rapid emergency-response production in plants of potentially pandemic influenza vaccines by Medicago Inc, among others (reviewed here: [35]). We see our future role in exploiting niche opportunities for
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production of vaccine candidates and reagents for orphan or geographically-limited disease agents that do not attract Big Pharma attention – like CCHFV and RVFV – as well as for emerging animal diseases such as BTV and AHSV and BFDV, where rapid responses and small manufacturing runs may be needed. We are still only near the beginning of our chosen road: working in a developing country for my whole career has meant facing challenges not experienced by more fortunate colleagues in the Global North, not the least of which are funding constraints and a lack of facilities for cGMP manufacture of APIs. However, I and others – including members of our international network, such as Rainer Fischer, Julian Ma and Charles Arntzen – seem to have influenced local stakeholders and local and even international funders of the exciting potential of molecular farming for transforming human and animal health care in developing country settings36 – and we look forward to an exciting destination at the end of our less-travelled road. References 1. Rybicki EP: Maize Streak Virus – an African Pathogen Come Home. S Afr J Sci 1988, 84(1):30-32. 2. Williamson AL, Rybicki EP: Detection of Genital Human Papillomaviruses by Polymerase Chain-Reaction Amplification with Degenerate Nested Primers. J Med Virol 1991, 33(3):165-171. 3. Williamson AL, Brink NS, Dehaeck CMC, Ovens S, Soeters R, Rybicki EP: Typing of Human Papillomaviruses in Cervical-Carcinoma Biopsies from Cape-Town. J Med Virol 1994, 43(3):231-237. 4. Ramesar JE, Rybicki EP, Williamson AL: Sequence Variation in the L1 Gene of Human Papillomavirns Type-16 from Africa. Arch Virol 1995, 140(10):1863-1870. 5. van Harmelen J, Wood R, Lambrick M, Rybicki EP, Williamson AL, Williamson C: An association between HIV-1 subtypes and mode of transmission in Cape Town, South Africa. Aids 1997, 11(1):81-87. 6. Hackland AF, Coetzer CT, Rybicki EP, Thomson JA: Genetically engineered resistance in tobacco against South African strains of tobacco necrosis and cucumber mosaic viruses. S Afr J Sci 2000, 96(1):33-38. 7. Palmer KE, Rybicki EP: The use of geminiviruses in biotechnology and plant molecular biology, with particular focus on Mastreviruses. Plant Sci 1997, 129 (2):115-130. 8. Palmer KE, Thomson JA, Rybicki EP: Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors. Arch Virol 1999, 144(7):1345-1360. 9. Palmer KE, Rybicki EP: Investigation of the potential of Maize streak virus to act as an infectious gene vector in maize plants. Arch Virol 2001, 146(6):1089-1104. 10. Varsani A, Williamson AL, Rose RC, Jaffer M, Rybicki EP: Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi. Arch Virol 2003, 148(9):1771-1786. 11. Kohl T, Hitzeroth II, Stewart D, Varsani A, Govan VA, Christensen ND, Williamson AL, Rybicki EP:
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Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-ofconcept study. Clin Vaccine Immunol 2006, 13 (8):845-853. Varsani A, Williamson AL, Stewart D, Rybicki EP: Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector. Virus Res 2006, 120 (1–2):91-96. Varsani A, Williamson AL, de Villiers D, Becker I, Christensen ND, Rybicki EP: Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16. J Virol 2003, 77(15):8386-8393. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson AL, Rybicki EP: Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 2007, 88:1460-1469. Jaffray A, Shephard E, van Harmelen J, Williamson C, Williamson AL, Rybicki EP: Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice. J Gen Virol 2004, 85:409-413. Halsey RJ, Tanzer FL, Meyers A, Pillay S, Lynch A, Shephard E, Williamson AL, Rybicki EP: Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles. Virus Res 2008, 133(2):259-268. Pillay S, Meyers A, Williamson AL, Rybicki EP: Optimization of Chimeric HIV-1 Virus-Like Particle Production in a Baculovirus-Insect Cell Expression System. Biotechnol Progr 2009, 25(4):1153-1160. Lynch A, Meyers AE, Williamson AL, Rybicki EP: Stability studies of HIV-1 Pr55(gag) virus-like particles made in insect cells after storage in various formulation media. Virol J 2012, 9.
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19. Chege GK, Shephard EG, Meyers A, van Harmelen J, Williamson C, Lynch A, Gray CM, Rybicki EP, Williamson AL: HIV-1 subtype CPr55(gag) virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine. J Gen Virol 2008, 89:2214-2227. 20. Chege GK, Burgers WA, Stutz H, Meyers AE, Chapman R, Kiravu A, Bunjun R, Shephard EG, Jacobs WR, Rybicki EP et al: Robust Immunity to an Auxotrophic Mycobacterium bovis BCG-VLP Prime-Boost HIV Vaccine Candidate in a Nonhuman Primate Model. J Virol 2013, 87(9):5151-5160. 21. Rybicki EP, Williamson AL, Meyers A, Hitzeroth II: Vaccine farming in Cape Town. Hum Vaccines 2011, 7 (3):339-348. 22. Meyers A, Chakauya E, Shephard E, Tanzer FL, Maclean J, Lynch A, Williamson AL, Rybicki EP: Expression of HIV-1 antigens in plants as potential subunit vaccines. Bmc Biotechnol 2008, 8. 23. Tanzer FL, Shephard EG, Palmer KE, Burger M, Williamson AL, Rybicki EP: The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine. Virol J 2011, 8. 24. Burgers WA, Chege GK, Muller TL, van Harmelen JH, Khoury G, Shephard EG, Gray CM, Williamson C, Williamson AL: Broad, high-magnitude and multifunctional CD4C and CD8C T-cell responses elicited by a DNA and modified vaccinia Ankara vaccine containing human immunodeficiency virus type 1 subtype C genes in baboons. J Gen Virol 2009, 90(Pt 2):468-480. 25. Burgers WA, Shephard E, Monroe JE, Greenhalgh T, Binder A, Hurter E, Van Harmelen JH, Williamson C, Williamson AL: Construction, characterization, and immunogenicity of a multigene modified vaccinia Ankara (MVA) vaccine based on HIV type 1 subtype C. AIDS Res Hum Retroviruses 2008, 24(2):195-206. 26. Mortimer E, Maclean JM, Mbewana S, Buys A, Williamson AL, Hitzeroth II, Rybicki EP: Setting up a platform for plant-based influenza virus vaccine production in South Africa. Bmc Biotechnol 2012, 12.
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27. Mortimer E, Hitzeroth II, Buys A, Mbewana S, Rybicki EP: An H5N1 influenza DNA vaccine for South Africa. S Afr J Sci 2013, 109(9-10). 28. Thuenemann EC, Meyers AE, Verwey J, Rybicki EP, Lomonossoff GP: A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J 2013, 11(7):839-846. 29. Pineo CB, Hitzeroth II, Rybicki EP: Immunogenic assessment of plant-produced human papillomavirus type 16 L1/L2 chimaeras. Plant Biotechnol J 2013, 11 (8):964-975. 30. Whitehead M, Ohlschlager P, Almajhdi FN, Alloza L, Marzabal P, Meyers AE, Hitzeroth II, Rybicki EP: Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice. Bmc Cancer 2014, 14. 31. Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP: High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 2010, 8 (1):38-46. 32. Duvenage L, Hitzeroth II, Meyers AE, Rybicki EP: Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastinlike polypeptides. J Virol Methods 2013, 191(1):55-62. 33. Regnard GL, Boyes RS, Martin RO, Hitzeroth II, Rybicki EP: Beak and feather disease viruses circulating in Cape parrots (Poicepahlus robustus) in South Africa. Arch Virol 2015, 160(1):47-54. 34. Walwyn DR, Huddy SM, Rybicki EP: Techno-Economic Analysis of Horseradish Peroxidase Production Using a Transient Expression System in Nicotiana benthamiana. Appl Biochem Biotech 2015, 175(2):841-854. 35. Rybicki EP: Plant-based vaccines against viruses. Virol J 2014, 11. 36. Rybicki EP, Hitzeroth II, Meyers A, Santos MJD, Wigdorovitz A: Developing Country Applications of Molecular Farming: Case Studies in South Africa and Argentina. Curr Pharm Design 2013, 19 (31):5612-5621.
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From plant virology to vaccinology: The road less travelled Edward Rybicki* Biopharming Research Unit; Department of Molecular & Cell Biology and Institute of Infectious Disease and Molecular Medicine; University of Cape Town; Cape Town, South Africa
*Correspondence to: Edward Rybicki; Email: ed. [email protected] http://dx.doi.org/10.1080/21645515.2015.1092751 www.tandfonline.com
The beginning of my interest in science was when I became fascinated with chemistry at an early age, by helping test the pH of my primary school’s swimming pool: as a child in the Zambia of the mid-1960s, this was quite an important consideration, as I spent a large part of my leisure time in it! I went on to develop a keen interest in medical and other sciences, by way of voracious reading: our little library at home was soon exhausted, but the Lusaka Public Library held me for a while. An early exposure to science fiction by way of a family friend also started a passion that has not yet waned – and further exposure to science, by way of Isaac Asimov, Poul Anderson, Robert Heinlein, Arthur C Clarke and other “hard science” exponents of SF. Senior school in England, Zambia and finally Zimbabwe cemented my desire to be a scientist, with an exposure to mathematics, physics, chemistry and biology at A-level – but it was biology that eventually won, due to the fact it was simply so much more interesting as a fact-rich environment, and biochemistry in particular. I took that interest to the University of Cape Town in 1974, where I quickly learned that I couldn’t do medicine or biology as I had registered too late, but that I could do botany. After an excruciatingly boring year during which I repeated much of my last year of school, doing maths, physics and chemistry, and managed to avoid learning too much about floral plant diversity or dissection, I discovered that now I could do medicine if I wanted to. I declined – I still hope this wasn’t a mistake – as biochemistry and chemistry were now beckoning, with microbiology as filler. Chemistry taught me an appreciation for stereochemistry, mechanism and molecular structure; biochemistry cemented the structure and molecule-as-mechanism Human Vaccines & Immunotherapeutics
basics – but surprisingly it was microbiology that really appealed to me, with glimpses of the different kinds of biology that could result from integrating the same sorts of molecules. This derailed my career plans somewhat, as I was locked into majoring in chemistry – and it now looked like I was going to have to do three majors, if I was to continue with microbiology. It ended up being no contest: given bacteriology with a whiff of immunology and virology in my first semester, I happily tossed biochemistry – and I have never looked back. I was also fortunate in having some inspirational teachers, one of whom – Marc van Regenmortel – I am still in professional contact with.
Plant Virology phase (1977 – 1987) I specialised in Virology with a plant virus serology project in my Honours degree in 1977 – a separate 1-year degree in South Africa, during which I tossed biochemistry again. I did another plant virology and serology project for a Masters degree from 1978 – 1979, during which I was introduced to advanced physical analytics of the bromoviruses, including by means of analytical ultracentrifugation and electrophoresis, and also to the then very novel ELISA techniques – techniques that still serve me well today. I then branched out into a more general molecular plant virology project for a PhD (1980 – 1984), in which I explored the purification and characterisation of vanishingly small amounts of barley yellow dwarf and other viruses from wheat and barley plants, and the use of western blotting as 2517
About Edward Rybicki Dr. Rybicki received his pre- and postgraduate education from the University of Cape Town, South Africa, where he has worked throughout his career, gaining experience as a visiting scientist at Plant Genetic Systems, Ghent, Belgium (1984), Boyce Thompson Institute, New York, U.S. (1990–1) and Arizona Biodesign Institute, U.S. (2010). He became a full Professor in Microbiology in 2003, was a founder member of the Institute of Infectious Disease and Molecular Medicine in the Faculty of Health Sciences, and is currently Director of the Biopharming Research Unit in the Molecular & Cell Biology Department at the University of Cape Town. His research originally focused on molecular characterization of plant viruses, which brought about pioneering discoveries in recombinant gene expression in plants and in insect cells. Dr. Rybicki developed these “biopharming” techniques to produce candidate vaccines against HPV-16, HIV-1 subtype C, influenza H5N1, and other viruses. He has authored >120 papers and 47 patents. He is a recipient of numerous awards and honors, including the President’s Award of the South African Foundation for Research Development (1985) and the Meiring Naude Medal of the Royal Society of South Africa (1986).
an analytical tool – also skills that were to serve me very well later – as well as accidentally co-discovering a novel aphid virus. I stayed at the University of Cape Town (UCT) despite the chance of going to the US for a PhD, because I got married to a UCT postgraduate student and got appointed to a Lectureship in Virology at UCT in 1981, before I had finished my PhD. I ended up effectively doing a postdoc in my own lab, as the award of my PhD coincided with my first major research grant, the so-called President’s Award from the local Foundation for Research Development. This allowed me to retool my expertise with a short sabbatical in a biotech company in Ghent, Belgium (Plant Genetic Systems) in order to learn the ins and outs of DNA cloning. I continued with my serological interests, however, including helping apply ELISA to Streptomyces taxonomy, applying western blot techniques to the affinity purification of monospecific antibodies, and discovering pathogenesis-related PR-1-type proteins in maize and other plants by western blotting.
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Molecular Plant Virology and PCR phase (1988 – 1996) In 1988, and on the strength of my newly acquired DNA manipulation skills, I staked a bold but in retrospect rather na€ıve claim on a plant virus – in the complete absence of any published work from our group on it. I wrote a review1 on Maize streak virus (MSV): this is a small single-stranded DNA plant virus that was discovered and largely described from Africa over some 70 years, and I was, in my mind at least, claiming it back from foreign domination. In fact, it would take my group and my colleague Ralph Kirby only another year to characterize three novel strains of the virus, and to apply restriction map-based phylogenetic analysis techniques to determining their evolutionary relationships with other MSVs. At the same time, I was collaborating with my ex-PhD supervisor Barbara von Wechmar and our PhD students on molecular characterisation of aphid picorna-like viruses, and plant potyviruses. In 1990 I did a year-long sabbatical at the Boyce Thompson Institute for Plant
Human Vaccines & Immunotherapeutics
Research in Ithaca, NY, with Steve Howell – and significantly expanded both my professional network and my skills base, with a nucleic acid-intensive set of projects largely based on the then very new polymerase chain reaction (PCR) technique that I had just started using. My geminivirus work evolved as my group and its molecular expertise grew, with characterisations of new potyviruses and geminiviruses by cloning and sequencing, and applications of PCR to detection and characterisation of diverse potyviruses and cereal-infecting geminiviruses – and also human papillomaviruses, with my recently-acquired medical virologist wife Anna-Lise Williamson.2-4 This work led to my developing a sideline in studying virus evolution, with major phylogenetic analyses published on both potyviruses and geminiviruses – and some collaboration on HIV-1 variation, with my former PhD student Carolyn Williamson.5 In a development that proved highly important for later work, I added a plant biotechnology string to my bow in this period, with supervision of projects involving making virus-resistant transgenic plants.
The Plant Biotechnology phase (1997 – 2002) While I continued to work with an expanding group on the molecular biology of geminiviruses in this period, and on transgenic resistance to viruses in plants,6 I had also begun with my student Kenneth Palmer to look at using geminiviruses for “molecular farming”, or the production of high-value biologics in plants.7 Work on MSV as an expression vector in cultured maize cells was especially useful,8,9 as it laid important groundwork for later molecular farming developments with another geminivirus. In particular, we were able to show stable, long-term (3 years) high-level expression of foreign genes in plant cells from plasmid-like circular dsDNA replicating forms of engineered MSV genomes. I was also prompted by Anna-Lise to “. . .express something useful!” when looking for a candidate gene to express in tobacco
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plants for molecular farming purposes – and we chose the HPV-16 L1 major capsid protein, both for reasons of familiarity due to our PCR work, and because we had the idealistic notion that we could make a cheap vaccine against cervical cancer in competition with Merck and GSK. During this time my lab also established a capacity to produce recombinant proteins in insect cells via engineered baculoviruses – ironically, as a means of providing “gold standard” geminivirus non-structural proteins for looking at MSV molecular biology.
The Early Vaccinology phase (2000 – 2009) Anna-Lise and I had received major local funding in 2000 to work on two completely different sets of vaccines: one grant, from the newly-formed South African AIDS Vaccine Initiative (SAAVI), was for the development of novel vaccines for South African variants of HIV-1 subtype C; the other, from the National Research Foundation (NRF)’s Innovation Fund, was for novel HPV vaccines. This stemmed from an idealistic desire on our behalf to try to make novel and affordable vaccines for Africa, given the complete absence of any manufacturing capacity in South Africa, as we had a spectrum of platforms for production of candidate vaccines between us – and given the opportunity to apply for two grants, we did, in the belief that we might get one. We were very pleasantly surprised, therefore, to get both! The SAAVI grant was renewed twice, to end in 2009, and the first NRF Innovation Award was followed by another specifically on plant- and insectmade HPV vaccines, and then other awards from the Poliomyelitis Research Foundation (PRF, South Africa), the SA Medical Research Council and the Cancer Association of SA (CANSA). My part of both projects had both insect cell and plant production arms: with HIV our object was to make budded virus-like particles (VLPs) from expression of the HIV-1 subtype C Pr55Gag polyprotein, as a booster protein vaccine for priming vaccinations with a DNA- or recombinant poxvirus- or BCG-vectored
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polygene encoding a Gag-RT-Tat-Nef fusion protein. With HPV we wanted to make generic L1 VLPs in plants as a cheaper and potentially orallyadministrable vaccine, novel chimaeric L1:L2 fusion proteins in insect cells as a second-generation vaccine with less type specificity, and to express L1 protein in BCG as another approach to making a cheap vaccine. My HPV work was quite successful from the outset: for plant expression, my team – co-supervised most ably by Inga Hitzeroth – was among the first in the world to make HPV-16 L1 VLPs in transgenic plants10; the first to show proof of efficacy in a rabbit model for a plantproduced L1 vaccine – for cottontail rabbit papillomavirus, CRPV11 – and the first to use a rTMV-vectored transient expression approach for any papillomavirus L1 (CRPV and HPV).11,12 Arvind Varsani in my group also successfully investigated, via baculovirus expression in insect cells, various surface locations in the L1 protein of HPV-16 for their potential to display a L2 peptide (108–120, LVEETSFIDAGAP) known to elicit widely crossneutralising antibodies.13 James Maclean went on to show that Agrobacteriummediated transient expression of HPV-16 L1 in Nicotiana benthamiana could be very significantly enhanced by use of human codon optimisation and localization of the protein in chloroplasts.14 HIV Gag expression work in insect cells was also highly successful: we were possibly the first to make HIV-1 subtype C Pr55Gag VLPs via baculovirus expression in insect cells, and showed that use of a matched gag DNA vaccine / Pr55Gag prime/boost regime gave excellent cytotoxic T-lymphocyte responses to Gag.15 We also showed that it was possible to express as VLPs the whole HIV Gag-RTTat-Nef (Grttn) polyprotein that was the basis of the SAAVI DNA/MVA vaccines being developed at the same time16 – but that GagRT or Gag-Tat-Nef (GagTN) had better yields and gave more uniform particles. We developed Gag VLP production into a reliable and reproducible process, with considerable optimisation of cell culture and other parameters17,18 – which led to our supplying our partner lab with VLPs for a series of landmark prime/
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boost experiments, involving a gag DNA/ Gag VLP combination19 and a gag BCG/ Gag VLP combination in two baboon experiments,19,20 showing that the combination was highly immunogenic, and induced a broad and polyfunctional central memory T-cell response appropriate for the control of HIV infection. Plant production of HIV-1 Gagderived antigens was less successful: yields of whole Pr55Gag were very low despite all optimisation efforts, and the best Ann Meyers in my group could do was prove production of VLPs – in itself a world first21 – and show that a MA/CA (p17: p24) fusion protein had some potential as a plant-produced HIV antigen.22 Something that came out of this work as a sideline, but which is proving to be highly useful in our continuing studies, was the development by Fiona Tanzer of an enhanced expression/DNA vaccine vector: this was based on a short enhancer element derived from the ssDNA Porcine circovirus 1, which increases protein expression in transfected cell cultures at least tenfold, and allows similar reductions in dosage of DNA vaccine required to elicit Grttn responses in mice.23 This would potentially allow significant dosesparing with the DNA vaccines involved in the SAAVI vaccine project,24,25 which went to clinical trial in 2008. Another sideshow project that has ended up being highly productive was the exploration of the potential of plant expression to produce pandemic influenza vaccines. As a result of a conference held in Cape Town in 2005, where a WHO influenza expert warned us “When the pandemic comes, you in the developing countries will be on your own”, we applied for extraordinary funding from the PRF in SA to explore the possibility of making a pandemic flu virus vaccine in South Africa. We chose the highly pathogenic avian influenza virus A H5N1 type haemagglutinin (H5 HA) as a target, and James Maclean was again instrumental in designing and successful early testing of plant-made soluble and membrane-bound forms. Further funding from the PRF and the SA MRC allowed proof of principle that we could in fact produce flu virus vaccine candidates in South Africa – both as subunit protein26 and as DNA vaccines.27
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In retrospect, while these projects were impossibly ambitious and not a little na€ıve, we and our co-workers received a crash course in both research vaccinology and the handling of big projects that has been crucial for all our subsequent work. We were also able to establish stable and wellqualified teams of people, with a nucleus of senior scientists who have been around us for up to 15 years. Another very important lesson was that we should patent our discoveries: in my case, this has led to me and my co-workers having the largest patent portfolio at our institution, and the largest molecular biotechnology-related portfolio in Africa – most of them to do with vaccines (14C patent families). The development of a set of well-tried protocols around expression of novel antigens in a variety of systems21 has also been invaluable – especially when funding circumstances demanded that we change direction.
Molecular Farming (2009 – 2015) Attendance of the very stimulating biennial Plant-Based Vaccines and Antibodies (now Biologics as well) Conferences since 2005 has led to very productive networking opportunities for us – as well as excellent funding opportunities. My group was fortunate enough to be included in a successful EU Framework 7 project called PlaProVa – Plant-Produced Vaccines (http://www.plaprova.eu/)-led by George Lomonossoff from the John Innes Centre in the UK, which involved us working on the development of plantmade vaccines against the newly-emerging Bluetongue virus (BTV) of sheep in Europe,28 as well as on plant production of our already-established HPV L1:L2 chimaeric vaccine candidates.29 We furthered ideas gained from the PBVAB on therapeutic vaccines for established HPV infections and cervical cancer into a collaborative project with Era Biotech in Spain, which resulted in a patent on the use of a maize zein seed storage proteinderived peptide (Zera) that causes protein body formation to be used as an adjuvant, for a shuffled HPV E7-derived synthetic protein vaccine.30 A happy marriage of my old plant virus interests with molecular farming occurred
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with the use of a mild strain of a beaninfecting geminivirus we had previously characterised as a replicating expression vector for tobacco, delivered by Agrobacterium31: our previous experience with MSV allowed rapid development of a versatile and high-yielding vector system that has been a highly valuable addition to our armamentarium. These projects have led on to more opportunities: the Technology Innovation Agency (TIA) and government Department of Science and Technology (DST) in South Africa have just funded us to work on novel BTV vaccines as well as vaccines for its fellow emerging orbivirus African horsesickness virus (AHSV), both major causes for concern in southern Europe as global warming progresses. Medicago Inc – possibly the world’s leading molecular farming vaccine company right now – is funding us to work on next-generation plant-produced HPV vaccines, and has patented a plant-made human rotavirus partially based on work done by us. We receive SA MRC and PRF funding for “One Health”-type projects on CrimeanCongo haemorrhagic (CCHFV) and Rift Valley fever (RVFV) bunyaviruses, where we are exploring the use of plants to make reagents as well as vaccines for these disease agents. We continue, on sniffs of funding from all over including patent royalties, to try to make vaccines against Beak and feather disease circovirus (BFDV), a ssDNA pathogen of parrots that threatens an endangered local species.32,33 Finally, we have branched out into reagent-based molecular farming, with the transient production in tobacco of recombinant horseradish peroxidase as a speciality reagent.34
The Future The potential importance of molecular farming for human health has been underlined recently with the apparently successful use of plant-produced MAbs (ZMapp) against Ebola virus disease in West Africa, and the proof of large-scale and rapid emergency-response production in plants of potentially pandemic influenza vaccines by Medicago Inc, among others (reviewed here: [35]). We see our future role in exploiting niche opportunities for
Human Vaccines & Immunotherapeutics
production of vaccine candidates and reagents for orphan or geographically-limited disease agents that do not attract Big Pharma attention – like CCHFV and RVFV – as well as for emerging animal diseases such as BTV and AHSV and BFDV, where rapid responses and small manufacturing runs may be needed. We are still only near the beginning of our chosen road: working in a developing country for my whole career has meant facing challenges not experienced by more fortunate colleagues in the Global North, not the least of which are funding constraints and a lack of facilities for cGMP manufacture of APIs. However, I and others – including members of our international network, such as Rainer Fischer, Julian Ma and Charles Arntzen – seem to have influenced local stakeholders and local and even international funders of the exciting potential of molecular farming for transforming human and animal health care in developing country settings36 – and we look forward to an exciting destination at the end of our less-travelled road. References 1. Rybicki EP: Maize Streak Virus – an African Pathogen Come Home. S Afr J Sci 1988, 84(1):30-32. 2. Williamson AL, Rybicki EP: Detection of Genital Human Papillomaviruses by Polymerase Chain-Reaction Amplification with Degenerate Nested Primers. J Med Virol 1991, 33(3):165-171. 3. Williamson AL, Brink NS, Dehaeck CMC, Ovens S, Soeters R, Rybicki EP: Typing of Human Papillomaviruses in Cervical-Carcinoma Biopsies from Cape-Town. J Med Virol 1994, 43(3):231-237. 4. Ramesar JE, Rybicki EP, Williamson AL: Sequence Variation in the L1 Gene of Human Papillomavirns Type-16 from Africa. Arch Virol 1995, 140(10):1863-1870. 5. van Harmelen J, Wood R, Lambrick M, Rybicki EP, Williamson AL, Williamson C: An association between HIV-1 subtypes and mode of transmission in Cape Town, South Africa. Aids 1997, 11(1):81-87. 6. Hackland AF, Coetzer CT, Rybicki EP, Thomson JA: Genetically engineered resistance in tobacco against South African strains of tobacco necrosis and cucumber mosaic viruses. S Afr J Sci 2000, 96(1):33-38. 7. Palmer KE, Rybicki EP: The use of geminiviruses in biotechnology and plant molecular biology, with particular focus on Mastreviruses. Plant Sci 1997, 129 (2):115-130. 8. Palmer KE, Thomson JA, Rybicki EP: Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors. Arch Virol 1999, 144(7):1345-1360. 9. Palmer KE, Rybicki EP: Investigation of the potential of Maize streak virus to act as an infectious gene vector in maize plants. Arch Virol 2001, 146(6):1089-1104. 10. Varsani A, Williamson AL, Rose RC, Jaffer M, Rybicki EP: Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi. Arch Virol 2003, 148(9):1771-1786. 11. Kohl T, Hitzeroth II, Stewart D, Varsani A, Govan VA, Christensen ND, Williamson AL, Rybicki EP:
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15.
16.
17.
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Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-ofconcept study. Clin Vaccine Immunol 2006, 13 (8):845-853. Varsani A, Williamson AL, Stewart D, Rybicki EP: Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector. Virus Res 2006, 120 (1–2):91-96. Varsani A, Williamson AL, de Villiers D, Becker I, Christensen ND, Rybicki EP: Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16. J Virol 2003, 77(15):8386-8393. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson AL, Rybicki EP: Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 2007, 88:1460-1469. Jaffray A, Shephard E, van Harmelen J, Williamson C, Williamson AL, Rybicki EP: Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice. J Gen Virol 2004, 85:409-413. Halsey RJ, Tanzer FL, Meyers A, Pillay S, Lynch A, Shephard E, Williamson AL, Rybicki EP: Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles. Virus Res 2008, 133(2):259-268. Pillay S, Meyers A, Williamson AL, Rybicki EP: Optimization of Chimeric HIV-1 Virus-Like Particle Production in a Baculovirus-Insect Cell Expression System. Biotechnol Progr 2009, 25(4):1153-1160. Lynch A, Meyers AE, Williamson AL, Rybicki EP: Stability studies of HIV-1 Pr55(gag) virus-like particles made in insect cells after storage in various formulation media. Virol J 2012, 9.
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19. Chege GK, Shephard EG, Meyers A, van Harmelen J, Williamson C, Lynch A, Gray CM, Rybicki EP, Williamson AL: HIV-1 subtype CPr55(gag) virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine. J Gen Virol 2008, 89:2214-2227. 20. Chege GK, Burgers WA, Stutz H, Meyers AE, Chapman R, Kiravu A, Bunjun R, Shephard EG, Jacobs WR, Rybicki EP et al: Robust Immunity to an Auxotrophic Mycobacterium bovis BCG-VLP Prime-Boost HIV Vaccine Candidate in a Nonhuman Primate Model. J Virol 2013, 87(9):5151-5160. 21. Rybicki EP, Williamson AL, Meyers A, Hitzeroth II: Vaccine farming in Cape Town. Hum Vaccines 2011, 7 (3):339-348. 22. Meyers A, Chakauya E, Shephard E, Tanzer FL, Maclean J, Lynch A, Williamson AL, Rybicki EP: Expression of HIV-1 antigens in plants as potential subunit vaccines. Bmc Biotechnol 2008, 8. 23. Tanzer FL, Shephard EG, Palmer KE, Burger M, Williamson AL, Rybicki EP: The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine. Virol J 2011, 8. 24. Burgers WA, Chege GK, Muller TL, van Harmelen JH, Khoury G, Shephard EG, Gray CM, Williamson C, Williamson AL: Broad, high-magnitude and multifunctional CD4C and CD8C T-cell responses elicited by a DNA and modified vaccinia Ankara vaccine containing human immunodeficiency virus type 1 subtype C genes in baboons. J Gen Virol 2009, 90(Pt 2):468-480. 25. Burgers WA, Shephard E, Monroe JE, Greenhalgh T, Binder A, Hurter E, Van Harmelen JH, Williamson C, Williamson AL: Construction, characterization, and immunogenicity of a multigene modified vaccinia Ankara (MVA) vaccine based on HIV type 1 subtype C. AIDS Res Hum Retroviruses 2008, 24(2):195-206. 26. Mortimer E, Maclean JM, Mbewana S, Buys A, Williamson AL, Hitzeroth II, Rybicki EP: Setting up a platform for plant-based influenza virus vaccine production in South Africa. Bmc Biotechnol 2012, 12.
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27. Mortimer E, Hitzeroth II, Buys A, Mbewana S, Rybicki EP: An H5N1 influenza DNA vaccine for South Africa. S Afr J Sci 2013, 109(9-10). 28. Thuenemann EC, Meyers AE, Verwey J, Rybicki EP, Lomonossoff GP: A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J 2013, 11(7):839-846. 29. Pineo CB, Hitzeroth II, Rybicki EP: Immunogenic assessment of plant-produced human papillomavirus type 16 L1/L2 chimaeras. Plant Biotechnol J 2013, 11 (8):964-975. 30. Whitehead M, Ohlschlager P, Almajhdi FN, Alloza L, Marzabal P, Meyers AE, Hitzeroth II, Rybicki EP: Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice. Bmc Cancer 2014, 14. 31. Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP: High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 2010, 8 (1):38-46. 32. Duvenage L, Hitzeroth II, Meyers AE, Rybicki EP: Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastinlike polypeptides. J Virol Methods 2013, 191(1):55-62. 33. Regnard GL, Boyes RS, Martin RO, Hitzeroth II, Rybicki EP: Beak and feather disease viruses circulating in Cape parrots (Poicepahlus robustus) in South Africa. Arch Virol 2015, 160(1):47-54. 34. Walwyn DR, Huddy SM, Rybicki EP: Techno-Economic Analysis of Horseradish Peroxidase Production Using a Transient Expression System in Nicotiana benthamiana. Appl Biochem Biotech 2015, 175(2):841-854. 35. Rybicki EP: Plant-based vaccines against viruses. Virol J 2014, 11. 36. Rybicki EP, Hitzeroth II, Meyers A, Santos MJD, Wigdorovitz A: Developing Country Applications of Molecular Farming: Case Studies in South Africa and Argentina. Curr Pharm Design 2013, 19 (31):5612-5621.
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