Archive for the ‘biofarming’ Category

Virology Africa 2015: Update and Registration

19 August, 2015

REGISTRATION IS NOW OPEN – VIROLOGY AFRICA 2015

On behalf of the Institute of Infectious Disease and Molecular Medicine of the University of Cape Town and the Poliomyelitis Research Foundation, we are pleased to invite you to Virology Africa 2015 at the Cape Town Waterfront.

VENUE AND DATES:

The conference will run from Tuesday 1st – Thursday 3rd December 2015. The conference venue is the Radisson Blu Hotel with a magnificent view of the ocean. The hotel school next door will host the cocktail party on the Monday night 30th November and in keeping with Virology Africa tradition, the dinner venue is the Two Oceans Aquarium.

IMPORTANT DATES

Early Bird Registration closes – 30 September 2015
Abstract Submissions deadline – 30 September 2015

The ACADEMIC PROGRAMME will include plenary-type presentations from internationally recognised speakers. We wish to emphasise that this is intended as a general virology conference – which means we will welcome plant, human, animal and bacterial virology contributions. The venue will allow for parallel workshops of oral presentations. There will also be poster sessions. Senior students will be encouraged to present their research. We have sponsorship for students to attend the meeting and details will be announced later in the year.

A program outline has been added to the website

WORKSHOPS

Our preliminary programme includes two workshops.

There is a hands-on workshop on “Plant cell packs for transient expression: Innovating the field of molecular biopharming”, with the contact person being Dr Inga Hitzeroth – Inga.Hitzeroth@uct.ac.za. This workshop will run at UCT one day before the conference, 30th November, and a second day, 4th December, after the conference.

The second workshop is on “”Viromics for virus discovery and viral community analysis”. The workshop at UCT will be on 4 and 5 December with the contact person being Dr Tracy Meiring – tracy.meiring@uct.ac.za.

Some of the workshop presenters will be integrated into the conference programme but the practical components will be run at University of Cape Town. Separate applications are necessary for each workshop.

If you are prepared to fund an internationally recognised scientist to speak at the conference or if you wish to organise a specialist workshop as part of the conference, please contact
Anna-Lise Williamson or Ed Rybicki.

For any enquiries please contact
Miss Bridget Petersen/ Email: conference1@onscreenav.co.za or phone: +27 21 486 9111
Ms Deborah McTeer/Email: conference@onscreenav.co.za or +27 83 457 1975

Don’t fear GMOs – fear the hype!

31 July, 2015
I’m going to share a slightly disturbing exchange I just had with a dietician – because it shows that even well-educated people out there are buying into the anti-GMO frenzy.

And I will thank +Mary Mangan for pointing out some of the sites mentioned!

“Dear xxxx;

In answer to your statements and questions:

“For a while, I have had the suspicion that GMO foods might be related to the epidemic increase in allergies, worldwide”

Ummmm…there is no good evidence of a worldwide epidemic of allergies – like autism, there is better recognition of the state, rather than an increase in incidence.

“my two boys clinically react to GMO varients,”

WHICH variants?  Of what?  How do you know?  This is a dangerous path, and I have trod it with immunologists involved in this sort of research – the ONLY way you can say “it is due to a GM food” is if you have the EXACT equivalent that is NOT GM – and by that, I don’t mean “GM vs non-GM maize” – because that is not biologically equivalent unless you have the same exact variety.  The other way would be to isolate the proteins involved, and test them – which is not that difficult, and is something I have thought of doing, if only to settle this issue for once and for all.

I would strongly urge you…to look at the links I will list below: most people, and medics and non-plant scientists as well, really don’t understand what actually happens with modern GM.  What happens is that one or a very few genes are introduced into a plant, to make one or possibly two proteins – against the 40 000+ the plant already makes. The genetic modification is minimal compared to conventional or advanced breeding, which moves around whole chromosomes, and MUCH easier to track than use of irradiation, which is also used to change traits – and very often changes things you can’t see and therefore ignore, unlike GM techniques.

What is more, all of the changes induced in plants can be followed these days by techniques like whole genome sequencing and proteomics, so that we can genuinely put hand on heart and say “this is exactly equivalent to that, except for one protein”.  Seriously: the question of equivalence is no longer really up for discussion; it is subject to evidence – and I will point out that the standards expected for GM plants are FAR more stringent than for conventionally-bred plants, which may have far bigger changes in protein composition than any GM variety.

http://www.ottawacitizen.com/touch/story.html?id=8738060

http://www.sciencebasedmedicine.org/index.php/antivaccine-versus-anti-gmo-different-goals-same-methods/

http://www.scoop.it/t/virology-news/curate?q=GMOhttp://www.scoop.it/t/virology-news/curate?q=GMO

http://gmopundit.blogspot.com/2011/09/convetional-corn-is-genetically.html

http://www.healthnewsdigest.com/news/Food_and_Nutrition_690/What-You-Need-to-Know-About-GMOs-GM-Crops-and-the-Techniques-of-Modern-Biotechnology.shtml

http://www.forbes.com/sites/henrymiller/2015/07/16/the-dumbest-most-pretentious-article-ever-about-genetic-engineering/

I hope this is helpful!

Sincerely,

Ed”

The double life of a geminivirus: Bean yellow dwarf virus

29 May, 2015

Every now and then I solicit contributions for this site – but this one came without coercion, or even prompting!  I thank Romana for being so enthusiastic B-)

Romana Yanez

Biopharming Research Unit, UCT

All material copyright Romana Yanez and UCT

***

I want to tell the story of a geminivirus called Bean yellow dwarf virus that has two very distinct “lives”: one as a crop pest, infecting bean plants in South Africa and the other as a powerful molecular tool as a viral vector for recombinant protein expression in plants. As if each one of the “heads” of the twinned capsids had a life of its own. The dark side and the bright side. The yin and yang…

Introduction

Geminiviruses are small, single-stranded, circular DNA plant viruses, so called because each particle is composed of two partially assembled icosahedra joined to form a twinned capsid [1], [2]. They infect plants and are carried by insect vectors such as leafhoppers and whiteflies [2]. They are divided into seven genera: Mastrevirus, Bogomovirus, Topocuvirus, Curtovirus, Becurtovirus, Eragrovirus and Turncurtovirus; according to their genomic organization, the hosts they infect, the insect vectors by which they are transmitted and by genome-wide pairwise sequence identities [3].

Geminiviruses belonging to the genus Mastrevirus are all monopartite viruses with genome sizes between 2.5 and 3.0 kb. They have as vectors different species of leafhoppers. They infect mostly monocotyledonous plants: Maize streak virus (MSV) causes devastating crop losses in African countries, Wheat dwarf virus (WDV), but also infect dicotyledonous plants: Bean yellow dwarf virus (BeYDV), Tobacco yellow dwarf virus (TYDV) and Chickpea chlorosis virus (CpCV) [2], [4], [5].

In 1997 the production of French beans (Phaseolus vulgaris cv. Bonus) was severely reduced  in South Africa, mainly in the Northern Province and Mpumalanga District [4]. Plants presented symptoms similar to a TYDV infection, which at the time was the only mastrevirus described to infect dicotyledonous plants. These symptoms included stunted growth, brittle and leathery leaves, and leaf curling. Investigating the aetiology of this disease were Liu and co-workers. They determined it was a geminiviral infection by identifying virus-like particles (VLPs) with the characteristic twinned morphology. They then sequenced DNA samples of the virus and found it to be most closely related to TYDV, both, in nucleotide sequence (65% identity) and in genomic organization. It was similar enough to be placed under the genus Mastrevirus but distinct enough to be considered a different virus. They Then called it Bean yellow dwarf virus – BeYDV [4].

In 2007 a mild strain of BeYDV (BeYDV-m) was described by Halley-Stott et al. also isolated from P. vulgaris (cv. Top Crop). It was phylogenetically similar to BeYDV having 97% nucleotide sequence identity, but presented sufficient phenotypic differences to be a different BeYDV strain. It contained 81 nucleotide differences compared to the BeYDV type, most of which (63 changes) were found in regions of the genome that directly influenced its replication. Thus, BeYDV-m produced typical symptoms that were less severe and temporally delayed when compared to BeYDV type. The authors also suggested that P. vulgaris is not the BeYDV natural host since it is a non-indigenous plant in South Africa. Furthermore, since both strains of BeYDV were isolated in the same region, it was also suggested that their natural host may show very mild or no symptoms upon infection [6]. Subsequently BeYDV-m was renamed as Chickpea chlorotic dwarf virus (CpCDV) [3]. [However, we like BeYDV, so we’re going to keep called it that – Ed]

Molecular Characteristics and Life Cycle of BeYDV

The genome of BeYDV (Figure 1) is 2,561 nucleotides long with an organization similar to that of other mastreviruses and that replicates by rolling circle mechanism [4], [7]. Its genome is bidirectional, consisting of virion-sense open reading frames (ORFs) V1 and V2, and complementary-sense ORFs C1, C2, C3 and C4. Of these, only C3 and C4 are non-functional and non-conserved between the mastreviruses; although they are also present in TYDV [4], [8]. Within the complementary sense ORFs C1 and C2 an intron is found which is also conserved in other mastreviruses. Virion sense and complementary sense ORFs are separated by a long intergenic region (LIR) and a small intergenic region (SIR) [4]. Liu and co-workers described the functions of each component of the BeYDV genome by mutational analysis.

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

The LIR contains a bidirectional promoter to which host factors can bind and a stem-loop structure within the origin of replication (ori) which is required for initiation of rolling circle replication. A binding site and nicking site for the replication associated protein (Rep) are also found in this region. The SIR in turn contains a primer binding region for initiation of complementary strand synthesis as well as transcription termination elements [8]. These are the only two cis-acting elements required for BeYDV replication [8], [10].

The V1 ORF encodes for the movement protein (MP) which is associated with plasmodesmata and is important for systemic spread of the virus. It was found to be a symptom inducer as transgenic plants expressing V1 developed wild type-like infection symptoms. The putative pathogen associated molecular pattern recognized by the host plant may be within the first 17 N-terminus amino acids as plants infected with a mutant  developed wild type-like symptoms as well [8]. 

The V2 ORF encodes for the capsid protein (CP) which is important for viral movement as well and therefore for systemic infection. Thus, intracellular movement or trafficking of the viral DNA may require encapsidation. This was suggested since V2 mutants did not infect plants systemically and also, a basic domain on the N-terminal of the CP was identified which putatively binds to DNA or is involved in nuclear localization [8], [11].

From the genome of BeYDV, the complementary sense ORFs C1 and C2 are the most interesting for me. These encode two regulatory proteins involved in the replication of the virus: Rep and RepA. Their expression is regulated by alternate splicing, where spliced C1 and C2 (C1C2) mRNA is translated into Rep and unspliced C1 mRNA is translated into RepA [8]. 

Rep is responsible for initiating rolling circle replication by nicking the stem-loop structure at the ori, and for releasing nascent virion sense single stranded DNA and later ligating it to form circular ssDNA molecules [8], [12]. Rep is the only protein required for BeYDV replication, but in the presence of RepA the replication is more efficient [8], [10], [11].

RepA is a multi-regulatory protein only found in mastreviruses [2]. Even though both Rep and RepA, have a retinoblastoma related protein (RBR)-binding motif, LeuXCysXGlu, in BeYDV only RepA is able to bind to RBR proteins [10]. In mammalian cells, the retinoblastoma protein is a tumor repressor that binds to and inactivates the transcription factor E2F. By binding to RBR proteins, RepA is thought to disrupt this interaction and force the plant cell cycle into the S-phase – where DNA is replicated just before cell division. RepA is thus acting like other viruses’ oncogenic proteins, such as the human papillomavirus E7 protein and the adenovirus E1A protein. Thus, keeping conditions favorable for enhanced viral replication and proliferation [10], [11]. This could be seen when Hefferon and Dugdale mutated the RBR binding-motif of Rep and RepA to LeuXCysXGln. Only the RepA mutant showed significantly decreased replication. While the Rep mutant showed wild type-like replication [11].

Having in mind what I just described, one can picture the life cycle of BeYDV as follows:

A leafhopper (which has not been identified yet) carrying the virus infects a host plant – this will be a dicotyledonous plant such as P. vulgaris, from which it was originally isolated. The virus releases its ssDNA genome into the cytoplasm. The ssDNA enters the nucleus where host’s replication machinery synthesizes the complementary strand from the primer located in the SIR region, generating a replicative double stranded circular DNA intermediate. At this point the dsDNA serves as template for gene expression, from which Rep and RepA are expressed. RepA transactivates virion-sense gene expression and interferes with plant cell’s life cycle to produce S-phase conditions. Rep nicks the stem-loop structure located at the ori and binds to the 5’ end of the nicked strand. The 3’ end acts as a primer for the synthesis of a new virion-sense strand displacing the previous virion-sense strand. When this new strand is complete, the ori is regenerated and Rep nicks it again. Subsequent release and recircularization of the nascent virion-sense strand is also mediated by Rep. The process continues on the new circular ssDNA molecules as well. Only later, when the amount of CP is high enough, ssDNA molecules are encapsidated. The CP and MP then mediate systemic spread of the viral genome [2], [8]–[12]. When another leafhopper visits the infected plant, the virus is transferred to other plants and all starts again (Figure 2).

gv fig 2

Figure 2. The life cycle of BeYDV. Black circle, BeYDV ssDNA with the stem-loop structure. Black and green circle, BeYDV dsDNA replicative intermediate. Orange spheres, plant host’s replication machinery. Yellow spheres, Rep protein. Black line, nascent ssDNA during rolling circle replication. Purple sphere, RepA. Green sphere, plant retinoblastoma-related protein. Red spheres, BeYDV movement protein. Geminal structures, BeYDV capsid proteins. Modified from [13], [14].

Liu et al. (1997) and Halley-Stott et al. (2007) showed that BeYDV is able to infect other dicotyledonous plants besides P. vulgaris, such as: Nicotiana tabacum, N. benthamiana, Datura stramonium and Arabidopsis thaliana [4], [6]. It has also been isolated from chickpeas in Pakistan [15]. It was noted by Liu and co-workers that the intron of BeYDV (and TYDV) is not as AU-rich as intron sequences present in dicotyledonous plants, which suggested that these viruses had evolved from monocotyledonous-infecting ancestors [8]. Other thing that suggests that BeYDV (and TYDV) evolved from monocotyledonous-infecting mastreviruses is that they encode for two variants of the Rep protein while other geminiviruses infecting dicotyledonous plants encode for only one Rep protein from a continuous ORF [11].

BeYDV as a Powerful Molecular Tool

I have talked about the relatively dark side of BeYDV as a crop pest and plant cell cycle manipulator. Now I would like to introduce you to the other face of this geminivirus.

The importance of recombinant proteins in pharmaceutical, medical and research fields makes them highly demanded, which in turn requires the use efficient production systems [16], [17]. Plants provide a cheaper, faster, more efficient and highly scalable platform for the production of proteins compared to other methods [18], [19]. Vectors based on DNA viruses can be used to express complex proteins without the limitations and complexity faced by RNA viruses such as the need to use more than one virus construct, size constraint imposed on the insert and genomic instability [2], [20]. BeYDV and other geminiviruses have small and simple DNA genomes which can be rapidly amplified to very high copy numbers using mainly host factors and that can be easily manipulated. These features make them attractive viruses for the design of plant vectors for the expression of recombinant proteins [21]. BeYDV has been extensively explored as a molecular tool for the expression of mainly pharmaceutically relevant proteins, such as vaccines, antibodies and enzymes [9], [21]. And recently it has also been used as a means to deliver reagents into plant cells to genetically engineer them [22].

Hefferon and co-workers were one of the first to design a vector derived from BeYDV. They expressed a synthetic version of Staphylococcus enterotoxin B (SEB) in tobacco NT-1 cells. The synthetic SEB sequence was placed under the control of a Cauliflower mosaic virus (CaMV) 35S constitutive promoter and flanked by the cis acting BeYDV LIR and SIR. The Rep encoding gene was provided in trans from a separate construct and also constitutively expressed from the CaMV 35S promoter. Constructs were co-delivered into NT-1 cells by bombardment [11]. They obtained expression levels of ≈0.025 mg SEB / kg of NT-1 cells. They showed that expression of SEB could be enhanced by 20 times by supplying Rep in trans compared to when no Rep was supplied. Overall they showed that BeYDV-based replicon systems promised enhancement of recombinant protein expression in plants [23].

In a more deconstructed approach, Mor et al. (2003) designed a replicon system similar to that of Hefferon and Dugdale (2003) in which the BeYDV MP and CP genes were replaced by the gene of interest (GUS), controlled by CaMV 35S promoter and flanked by the LIR and SIR sequences [24]. Since the CP can sequestrate viral ssDNA, preventing dsDNA to be formed [8], by removing the CP from the viral vector, expression levels can be increased. Removing non-essential features of the virus also gives more room for larger inserts and channels energy and building blocks that would be used to synthesize these proteins into expressing the recombinant protein [20]. Mor et al. obtained expression levels 40 times higher when supplying Rep as well as RepA than when no Rep/RepA was supplied. Showing that RepA also enhances expression levels, probably by making the cell environment more favorable for replication [24]. 

Regnard et al. (2010) designed a replicon vector, pRIC, based on the mild strain of BeYDV that contained the Rep/RepA coding regions in cis rather than in trans. This allowed the vector to autonomously replicate and thus generate high levels of gene copy number and in turn enhanced protein expression. They used N. benthamiana plants and Agrobacterium tumefaciens-mediated gene delivery. They obtained higher expression levels than previously described of three unrelated proteins: enhanced GFP, Human Papillomavirus type 16 major CP, L1, and a HIV-1 p24 antigen. Yields were higher when using the replicative vector than when compared to expression from a non-replicative A. tumefaciens expression vector: 550 mg ⁄ kg fresh leaf weight (FLW) vs. 337 mg L1 ⁄ kg FLW for L1 and 3.23 mg p24 ⁄ kg FLW vs. 0.95 mg p24 ⁄ kg FLW for p24. This study showed that autonomous replication of BeYDV-based vectors dramatically increases gene expression levels [25].

Huang et al. (2009) designed a three-component replicon system that consisted of a construct derived from a deconstructed version BeYDV similar to that described by Mor et al. (2003) containing the gene of interest expression cassette, a construct encoding for the Rep/RepA under CaMV 35S promoter control and a construct expressing the posttranscriptional gene silencing suppressor protein P19. They obtained 0.34 g of Norwalk virus CP (NVCP) / kg FLW and 0.8 g of hepatitis B core antigen (HBc) / kg FLW, which were able to form VLPs. In order to simplify the replicon system, they included the Rep/RepA sequences in cis. They obtained similar expression levels when using the simplified replicon, with or without P19 supplementation as when the three-component system was used [26].  Later they designed a single vector containing multiple replicon cassettes each flanked by a LIR and a SIR. The vector also contained the Rep/RepA sequences under LIR control. Co-delivering the single-vector replicon and a P19 expression vector, they expressed the light and heavy chain of an Ebola virus-targeting monoclonal antibody (mAB), 6D8. They obtained ≈0.5 g of 6D8 mAB / kg FLW which had been assembled correctly and could bind its antigen specifically. Expression levels were comparable to those obtained by Giritch et al. (2006) [27] using two vectors based on two non-competing RNA viruses. They speculated that using this single-vector multireplicon system, even four proteins could be expressed simultaneously using two vectors or placing expression cassettes in tandem  [28].

More recently, Moon et al. (2014) were able to express Brome mosaic virus (BMV) and Cucumber mosaic virus (CMV) VLPs at 0.5 and 1.0 g / kg FLW respectively, using a BeYDV-derived single-vector replicon system. This vector included the P19 coding sequence, the gene of interest as well as the Rep/RepA coding sequences in the same backbone. In this way enhanced expression of VLPs that can be used as carriers for nano-platforms with applications in material sciences and medicine was possible with only one agroinfiltration [29].

Finally, Baltes et al. (2014) demonstrated that BeYDV-based replicon system can be also used for plant genome engineering. They were able to deliver various nucleases (TALENs and CRISP/Cas system) as well as repair templates into tobacco cells and to regenerate plantlets with the desired DNA changes within 6 weeks. This highlighted the potential of vectors derived from BeYDV and other geminiviruses to be applied in the engineering of plants for, for example,  improvement of crop characteristics, crop resistance or in fundamental biology studies [22].

In conclusion, BeYDV is a small, dicotyledonous plant-infecting mastrevirus with apparently unlimited possible molecular applications.

References

[1] W. Zhang, N. H. Olson, T. S. Baker, L. Faulkner, M. Agbandje-McKenna, M. I. Boulton, J. W. Davies, and R. McKenna, “Structure of the Maize streak virus geminate particle.,” Virology, vol. 279, pp. 471–477, 2001.

[2] E. P. Rybicki and D. P. Martin, “Virus-derived ssDNA vectors for the expression of foreign proteins in plants,” Current Topics in Microbiology and Immunology, vol. 375, pp. 19–45, 2011.

[3] A. Varsani, J. Navas-Castillo, E. Moriones, C. Hernández-Zepeda, A. Idris, J. K. Brown, F. Murilo Zerbini, and D. P. Martin, “Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus,” Archives of Virology, vol. 159, pp. 2193–2203, 2014.

[4] L. Liu, T. Van Tonder, G. Pietersen, J. W. Davies, and J. Stanley, “Molecular characterization of a subgroup I geminivirus from a legume in South Africa,” Journal of General Virology, vol. 78, pp. 2113–2117, 1997.

[5] J. Hadfield, J. E. Thomas, M. W. Schwinghamer, S. Kraberger, D. Stainton, A. Dayaram, J. N. Parry, D. Pande, D. P. Martin, and A. Varsani, “Molecular characterisation of dicot-infecting mastreviruses from Australia,” Virus Research, vol. 166, no. 1–2, pp. 13–22, 2012.

[6] R. P. Halley-Stott, F. Tanzer, D. P. Martin, and E. P. Rybicki, “The complete nucleotide sequence of a mild strain of Bean yellow dwarf virus,” Archives of Virology, vol. 152, pp. 1237–1240, 2007.

[7] K. E. Palmer and E. P. Rybicki, “The molecular biology of mastreviruses.,” Advances in virus research, vol. 50, pp. 183–234, 1998.

[8] L. Liu, J. W. Davies, and J. Stanley, “Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants,” Journal of General Virology, vol. 79, pp. 2265–2274, 1998.

[9] Q. Chen, J. He, W. Phoolcharoen, and H. S. Mason, “Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants,” Human Vaccines, vol. 7, no. 3, pp. 331–338, Mar. 2011.

[10] L. Liu, K. Saunders, C. arole L. Thomas, J. W. Davies, and J. Stanley, “Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.,” Virology, vol. 256, pp. 270–279, 1999.

[11] K. L. Hefferon and B. Dugdale, “Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication,” Journal of General Virology, vol. 84, pp. 3465–3472, 2003.

[12] C. Gutierrez, “Geminivirus DNA replication,” Cellular and Molecular Life Sciences, vol. 56. pp. 313–329, 1999.

[13] “Adult drawing grape leafhopper,” Koppert Biological Systems 9103. [Online]. Available: https://www.flickr.com/photos/koppert/2400156751/. [Accessed: 09-Feb-2015].

[14] “Phaseolus vulgaris,” Belgium, Prelude – Royal Museum for Central Africa – Tervuren. [Online]. Available: http://www.africamuseum.be/collections/external/prelude/view_plant?pi=09910. [Accessed: 09-Feb-2015].

[15] N. Nahid, I. Amin, S. Mansoor, E. P. Rybicki, E. Van Der Walt, and R. W. Briddon, “Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan,” Archives of Virology, vol. 153, pp. 1441–1451, 2008.

[16] G. Pogue and S. Holzberg, “Transient Virus Expression Systems for Recombinant Protein Expression in Dicot-and Monocotyledonous Plants,” in Plant Science, N. K. Dhal and S. C. Sahu, Eds. InTech, 2012, pp. 191–216.

[17] F. Sainsbury, P.-O. Lavoie, M.-A. D’Aoust, L.-P. Vézina, and G. P. Lomonossoff, “Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2.,” Plant biotechnology journal, vol. 6, no. 1, pp. 82–92, Jan. 2008.

[18] E. P. Rybicki, “Plant-produced vaccines: promise and reality.,” Drug Discovery Today, vol. 14, no. 1–2, pp. 16–24, Jan. 2009.

[19] V. Yusibov, S. Rabindran, U. Commandeur, R. M. Twyman, and R. Fischer, “The potential of plant virus vectors for vaccine production.,” Drugs in R&D, vol. 7, no. 4, pp. 203–17, Jan. 2006.

[20] Y. Gleba, S. Marillonnet, and V. Klimyuk, “Plant Virus Vectors: Gene Expression Systems,” Encyclopedia of Virology, vol. 4, pp. 229–237, Apr. 2008.

[21] K. L. Hefferon, “DNA Virus Vectors for Vaccine Production in Plants: Spotlight on Geminiviruses,” Vaccines, vol. 2, no. 3, pp. 642–653, Aug. 2014.

[22] N. J. Baltes, J. Gil-Humanes, T. Cermak, P. a Atkins, and D. F. Voytas, “DNA replicons for plant genome engineering.,” The Plant cell, vol. 26, no. January, pp. 151–63, 2014.

[23] K. L. Hefferon and Y. Fan, “Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system,” Vaccine, vol. 23, pp. 404–410, 2004.

[24] T. S. Mor, Y.-S. Moon, K. E. Palmer, and H. S. Mason, “Geminivirus vectors for high-level expression of foreign proteins in plant cells.,” Biotechnology and bioengineering, vol. 81, pp. 430–437, 2003.

[25] G. L. Regnard, R. P. Halley-Stott, F. L. Tanzer, I. I. Hitzeroth, and E. P. Rybicki, “High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector.,” Plant biotechnology journal, vol. 8, no. 1, pp. 38–46, Jan. 2010.

[26] Z. Huang, Q. Chen, B. Hjelm, C. Arntzen, and H. Mason, “A DNA replicon system for rapid high-level production of virus-like particles in plants.,” Biotechnology and bioengineering, vol. 103, no. 4, pp. 706–14, Jul. 2009.

[27] A. Giritch, S. Marillonnet, C. Engler, G. van Eldik, J. Botterman, V. Klimyuk, and Y. Gleba, “Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 40, pp. 14701–6, Oct. 2006.

[28] Z. Huang, W. Phoolcharoen, H. Lai, K. Piensook, G. Cardineau, L. Zeitlin, K. J. Whaley, C. J. Arntzen, H. S. Mason, and Q. Chen, “High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system.,” Biotechnology and bioengineering, vol. 106, no. 1, pp. 9–17, May 2010.

[29] K. Moon, J. Lee, S. Kang, M. Kim, H. S. Mason, J. Jeon, and H. Kim, “Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.,” Applied microbiology and biotechnology, vol. 98, pp. 8281–90, 2014.

Ethical dilemma for Ebola drug trials

13 November, 2014

Public-health officials split on use of control groups in tests of experimental treatments.

With clinical trials of experimental Ebola treatments set to begin in December, public-health officials face a major ethical quandary: should some participants be placed in a control group that receives only standard symptomatic treatment, despite a mortality rate of around 70% for Ebola in West Africa?

Two groups planning trials in Guinea and Liberia are diverging on this point, and key decisions for both are likely to come this week. US researchers meet on 11 November at the National Institutes of Health (NIH) in Bethesda, Maryland, to discuss US-government sponsored trials. A separate group is gathering at the World Health Organization (WHO) in Geneva, Switzerland, on 11 and 12 November to confer on both the US effort and trials organized by the WHO with help from African and European researchers and funded by the Wellcome Trust and the European Union.

Source: www.nature.com

I have to say – faced with a deadly disease, I think it is UNethical to have control / placebo arms of any trial.

Seriously: what about comparing ZMapp and immune serum, for example, with historical records of previous standard of care outcomes rather than directly?

I know if I were an Ebola patient, and I saw someone else getting the experimental therapy and I didn’t, that I would have a few things to say.

It’s not as if these therapies have not been tested in primates, after all – in fact, both the ChAd3 and MVA-based vaccines and ZMapp have been thoroughly tested in macaques, as have the other therapeutics, with no adverse events there.

I say if people say clearly that they want an experimental intervention, that they should get one: after all, the first use of immune serum was not done in a clinical trial, but rather as a last-ditch let’s-see-if-this-works intervention – yet its use does not seem controversial?

See on Scoop.itVirology News

Virology Africa 2015: consider yourselves notified!

7 November, 2014

Dear ViroBlogy and Virology News followers:

Anna-Lise Williamson and I plan to have another in our irregular series of “Virology Africa” conferences in November-December 2015, in Cape Town.

As previously, the conference will run over 3 days or so, possibly with associated workshops, and while the venue is not decided, we would like to base it at least partially in the Victoria & Alfred Waterfront.

We also intend to cover the whole spectrum of virology, from human through animal to plant; clinical aspects and biotechnology.

We intend to make it as cheap as possible so that students can come. We will also not be inviting a slate of international speakers, as we have found that we always get quite an impressive slate without having to fund them fully.

It is also the intention to have a Plant Molecular Farming workshop – concentrating on plant-made vaccines – concurrently with the conference, in order to leverage existing bilateral travel grants with international partners. If anyone else has such grants that could be similarly leveraged, it would be greatly appreciated.

See you in Cape Town in 2015!

Ed + Anna-Lise

ZMapp in an HIV context

30 October, 2014

It was truly a pleasure to run into Kevin Whaley of Mapp BioPharmaceutical today, here at the HIVR4P inernational conferrence in Cape Town – so I made him come and have coffee with me and Anna-Lise, so we could chat about molecular farming.
Of course, it is the ZMapp plant-made therapeutic antibody that has set the molecular farming world alight, that was the main topic. Apparently Mapp is looking at a January 2015 date for a clinical trial in the affected West African countries, alongside the adenovirus and RSV-vectored vaccines. The plants for the production of the thousands of doses that will be needed – and recall, that’s a couple of grams per dose at 50 mg/kg – are already growing at Kentucky Bioprocessing in Louisville, so one imagines that a pile of work will be coming their way in the near future.
It’s also sobering to realise that even though plants ARE a more scalable and POTENTIALLY cheaper means of production of biologics, that therapeutic antibody production in particular, MAY be better suited right now to conventional technologies, such as CHO cell or even fungal production.
This is because large quantities of MAbs will be needed, and there is established capacity for production of hundreds of thousands of litres of cell culture right now, and yields and production costs have been driven right down to US$10 / gram for MAbs already, according to Kevin.
This partly answers a question I had during the HIVR4P sessions: if one is to use 20-50 mg/kg dosages for anti-HIV neutralising MAbs such as VRC01, how would it be remotely possible to make the amounts required for use in a developing country setting, where the patient can almost definitely NOT pay?
I still think there is a role for plants – but maybe this will be in the area of prophylactic use of MAbs, where much lower doses may be effective because there is not nearly as much virus to neutralise or inactivate.
And of course, Mapp is involved here too, with plant-made VRC01 in particular being incorporated into microbicides.
A great bunch of people, with really noble aims.

Norway to get world’s last dose of ZMapp – update

8 October, 2014

The Norwegian woman, infected by the Ebola in Sierra Leone and currently receiving treatment in Oslo, will get the last dose of the virus treatment medicine ZMapp

Source: m.thelocal.no

…and yet again, the emphasis is on how slow it is to make it – when the whole point of biofarming and transient expression is that it is supposed to be QUICK to make things, and easy to scale up production!!

What is the problem here?  KBP has facilities – or says it does – for large-scale production of proteins via transient expression in N benthamiana via rTMV or even BeYDV-based vectors. SO why has it been so difficult to make more ZMapp??

Why, in fact, are we told via other reports that the US government is considering getting Caliber to make it, or even to make the cocktail in CHO cells, because of capacity, when KBP has the equipment?

It can’t be supply of plants, surely: if they’d planted out a big greenhouse or two of N benth the moment ZMapp hit the news, they’d have enough to make many grams of ZMapp right now – given that it takes just a few days of incubation post-infiltraiton to make the protein.

Surely it’s not a protein purification thing – because THAT’S pretty quick too, once the plants have been mushed.

So what IS the bottleneck? cGMP requirement? Lack of certified protocols / equipment? Can someone tell us??  Otherwise, a posterchild for biofarming will end up being made by good old stainless steel cell culture technology, and our favourite way of doing things will have been found to be wanting.

NOTE ADDED 10th October:

Never let it be said I was unwilling to get schooled by a former colleague…Kenneth Palmer just told me what the problem is:

“You may not be aware that the human dose of Zmapp is 12 grams per patient, 3 infusions of 4 grams each.  Check the dose in recent Nature paper. If yield of one antibody is 100 mg per kg and you have to produce three antibodies for Zmapp… If you do the arithmetic you will see why the process is “slow””.

So…. Doing just that, you end up with 30 kg N benthamiana per gm of ZMapp as a best-case yield – meaning 360 kg PER PATIENT.

That’s a LOT of N benth – and tooling up for that sort of plant production takes time. Thanks, Kenneth!

I would be VERY interested in a cost breakdown of ZMapp vs CHO cell-produced MAbs – because producing at that sort of scale MUST be prohibitively expensive in stainless steel?

 

See on Scoop.itPlant Molecular Farming

Engineering of inducible resistance in maize to Maize streak virus: a history, and a model for South-South collaboration

16 September, 2014
A Maize streak virus particle: characteristic doubled icosahedron containing a single ssDNA Graphic by Russell Kightley Media

A Maize streak virus particle: characteristic doubled icosahedron containing a single ssDNA
Graphic by Russell Kightley Media

I blogged on this paper from our group over at Virology News, but then I decided to do it again here. Because, as I said there,

“This is a big deal: seriously. It’s the culmination of some 24 years of involvement by my lab in engineering resistance in maize, and is the latest effort on top of of one unsuccessful and one partially successful construction by three top-class researchers in that time.”

…and I don’t think I did justice at the time to acknowledging the amount of effort it has taken to get to this point – which has occupied nearly 30 years of my life, and a considerable fraction of the working life of many others.  So here’s something of a chronicle of how we got here from there with some explanation of the very cool science behind it, and acknowledgement of a very valuable friendship between us and colleagues in Australia, without whom this would not have happened.

Maize streak virus is, of course, possibly the first virus described from Africa, and one famous enough to have its own web page, complete with the Proceedings of the First (and last) International Maize Streak Disease Symposium held in Hazyview in South Africa in 1997.  It is a ssDNA virus with a 2.7 kb circular genome that is encapsidated in unique geminate particles, is obligately transmitted by the leafhopper Cicadulina mbila Naude, and is probably the worst viral pathogen of maize in Africa.

The Maize streak virus investigations that led to this latest development were started by my retired colleague and former PhD supervisor Prof Barbara von Wechmar, back in the 1960s at the University of Stellenbosch.  Barbara it was who isolated MSV out of naturally infected maize, and developed home-grown methods of propagating it in sweetcorn via viruliferous Cicadulina mbila leafhoppers – and more importantly, getting clean leafhopper colonies so that new isolates could be studied.

She brought this knowledge with her to UCT when she came here with Marc van Regenmortel as the new Professor of Microbiology, and the leafhopper work and virus isolates puttered on in the background, with a couple of interesting papers coming out. The first was a collaborative work with the legendary Bob (RG) Milne, who came here on a sabbatical from the Istituto di Fitovirologia Applicata del CNR in Torino, Italy, and revolutionised our electron microscopy techniques by use of uranyl acetate instead of phosphotungstate, which meant for the first time we could see MSV particles.  This got presented at an International Maize Virus Disease Colloquium and Workshop in Wooster, Ohio in 1982, the Proceedings of which  seem to be available here.

VON WECHMAR, M.B., & MILNE, R.G. (1983). Purification and serology of a South African isolate of maize streak virus. pp. 164-166 In: Proc. Internat. Maize Virus Dis. Colloquium and Workshop. University of Ohio Press, Wooster

Maize streak virus particles isolated from maize, photographed by Robert G Milne in Cape Town

Maize streak virus particles isolated from maize, photographed by Robert G Milne in Cape Town

Another paper in J Gen Virol from 1983 was a fascinating account of using “electroinfection” to infect maize plants with MSV, published with Dr Alfred Polson: he was a quirky and idiosyncratic physical biochemist-cum-virologist who had done a PhD with The Svedberg and Ole Lamm in Uppsala in the 1930s, headed a Virus Research Unit at UCT Medical School, then retired to our Department to basically play with physical and serological techniques.  He liked nothing better than to hook plants up to kilovolt power packs, and to do large-scale electrophoresis of proteins and viruses in convoluted home-made glass contraptions – and to pull us in to help.

Also in the early 1980s, when molecular microbiology belatedly dawned in South Africa, a couple of DNA-wise colleagues and I thought it would be a great idea to sequence the MSV genome, given that it had been shown in 1977 in a Nature paper by Bryan Harrison and colleagues to be circular ssDNA.  Sadly, this never really got past the talking and wasting virus sample stage by Frank Robb and Ralph Kirby before two papers were published on the sequence of MSVs in 1984 – with the second, by Phil Mullineaux and colleagues from the John Innes Institute, drily pointing out that the first, by Steve Howell, had the sequence of the complementary, rather than the genomic strand of DNA.

I decided in 1985, after getting a PhD on a biophysical and serological investigations of small grain viruses in 1984, to change my skill set during a three-month academic leave in Belgium by learning molecular cloning and DNA handling techniques.  Thanks to the recommendation of Marc van Montagu, and the teachings of the good folks at Plant Genetic Systems in Gent (Jan Leemans and Herman Hofte, I still owe you B-), this was achieved – and the first thing I did on my return was to recruit an able Honours student in the person of Bev Clarke, cosupervised by Ralph Kirby for the DNA expertise, to clone and sequence local MSV strains that were still being maintained by Barbara von Wechmar.  Bev worked through into a Masters degree on cloning and restriction mapping of three maize isolates of MSV – sequencing was still a bit complicated and expensive in those days – and we managed to publish two babck-to-back papers out of her work, in 1989.  The first was an account of the propagation, isolation, cloning and mapping, and we in fact got the cover of that issue.

intervirol coverThe second was the start of what became a really interesting sideline for me, in evolutionary studies on viruses – because Ralph managed to use the maps we generated from our viruses and from sequenced isolates to estimate sequence divergence. It really is quite amusing, in this era of rapid and metagenomic sequencing, to read what we thought we had accomplished at the time, given that my team has just sequenced some ten geminiviruses as part of a BSc third year project:

“The aligned restriction endonuclease maps of three sequenced maize streak virus isolates, three restriction-mapped southern African maize streak virus isolates, and two other sequenced geminiviruses were used as a means of calculating the sequence divergence between these viruses. The degree of divergence was used to construct a phylogenetic tree for the viruses; this tree agrees well with predictions from sequence comparisons, and so the method can be used to study the relationship of geminivirus isolates without the labor and expense of sequencing each one. [my emphasis]”

On the strength of this and other work, I was prompted in 1988 to write a bold (and naive) lamppost-marking type of article on “Maize streak virus: an African pathogen come home?”, which I commemorated 25 years later here in ViroBlogy.  I cringed a little then and again now, seeing what we thought was so cool at the time. Ah, youth…B-)

Bev was succeeded as “the” student in my lab by Fiona Tanzer (then Hughes), who took over the MSV and other geminivirus work with verve and flair.  She produced a number of papers during her PhD, starting with an offering on “A rapid technique for typing streak virus isolates using a panel of differential hosts” at a South African Maize Breeding Symposium in 1990, and going on to characterise Sugarcane streak mastrevirus (SSV) from Natal as a distinct virus in 1991, by RE mapping, Southern hybridisation and partial sequence analysis; “Genome Typing of Southern African Subgroup-1 Geminiviruses” by the same techniques in 1992, and graduating to “Complete nucleotide sequence of sugarcane streak Monogeminivirus” in 1993.

Meantime, in what turned out to be my last stint of concentrated laboratory work, I had been investigating the potential of PCR for both detection and differentiation of mastreviruses – which led to a little detour into papillomavirology to apply the technique, which ended up being a complete change in direction, and a whole new career in vaccinology.  I used the primer design experience and PCR results I got then as the basis of an online teaching module on PCR, which is still available – and for which I actually get citations.

Degenerate Primer PCR for Mastrevirus Detection

But I digress: Fiona went on after her PhD to work as a postdoc with me and Jennifer Thomson, with whom I had been collaborating since she arrived at UCT in 1988 in the area of plant genetic engineering, on an insanely ambitious project to engineer transgenic resistance to MSV in maize.  This had in fact started in 1990, when I visited Bill Gordon-Kamm at DeKalb Genetics in Connecticut while on sabbatical at Cornell in 1990-1991: I got a suspension culture of Black Mexican Sweet (BSM) maize cells from them for us to practice biolistic techniques on with our newly-acquired BioRad helium gene gun, as well as a collaboration agreement which helped us immensely in our maize transformation and regeneration work.  Sandy Lennox it was who got that side of things working, to the extent of regenerating viable plants from supposedly non-regenerable BMS cells – which laid the foundation for all of our subsequent work in this area, using HiII embryogenic callus cultures.

Fiona, meanwhile, had managed to both clone and sequence a moderately severe isolate of MSV from Komatipoort (MSV-Kom), and then also make it agroinfectious as a partially dimeric clone in Agrobacterium tumefaciens, following the landmark 1987 example of Nigel Grimsley and colleagues in Basel.  This provided the other half of the toolkit, as we now had a means of reliably testing regenerated maize with a MSV isolate of known virulence, available for infection as a clone rather than transmitted via leafhoppers.

Fiona went on to make and test antisense RNA-expressing constructs from the Rep gene of MSV-Kom, as I had been heavily influenced by a 1991 Keystone Symposium on Antisense RNA Technology I went to while on sabbatical – and reported on here – and was convinced this was the way to go.

Inevitably, as determined by exhaustive experimentation from laboriously regenerated plants, this turned out not to be true: there was no obvious protection of the highly susceptible HiII by any of the constructs.  Fiona went off to have a baby – her second while working with me – and the MSV effort stalled for a while.  Barbara retired, and it fell to me to maintain leafhopper colonies and MSV and other mastrevirus isolates – which, very fortunately, we had begun routinely cloning and making agroinfectious, via a very able cohort of students led by Wendelin Heribert “Popeye” Schnippenkoetter, who perfected the art of the “1.1-mer” infectious clone as well as finally publishing the MSV-Kom sequence.

The value of international networking in geminivirology became apparent in 1994, while all this this was happening, when I went to the first International Geminivirus Symposium in Almeria, Spain.  There I re-made the acquaintance of  Doug Maxwell, who I had met in Ithaca in 1991 when he spoke on using PCR to detect begomoviruses.  I also recall performing “Born to be Wild” in a hotel karaoke bar with a scratch band of geminivirologists, but we shall speak no further of this.

gemtax_ppt

Doug and I corresponded until the next Symposium he organised in Puerto Rico in 1998, when we heard his PhD student Steve Hanson present on the use of trans-dominant Rep gene mutants to inhibit the replication of Bean golden mosaic begomovirus in transient assays in cultured bean cells.  They sent us the PhD thesis to use as a reference document; the work was eventually published in 1999.

This kicked our efforts into a higher gear, given that we now had an exemplar in another albeit VERY distantly related geminivirus.  We also had a very bright new PhD student from Zimbabwe, Tichaona Mangwende, as the perfect guinea pig for investigation of dominant negative Rep mutants, and off we set again.  Tich quickly made a number of site-directed mutants by very ingenious methods, and we then came up to the hurdle of how to test them.  We had had in mind that this would be done in regenerated plants; however, it was quickly apparent that this would stretch his project out for several years longer than was feasible.

Here it was that the value of having students working in related projects was shown: after 1996 I had received expanded funding due to a favourable Foundation for Research Development rating, and actually had a lab full of students all working on geminiviruses.  Fortuitously, Kenneth Palmer had been doing a project on exploring the potential of MSV-based constructs as recombinant expression vectors for use in maize, and had worked out a suite of techniques for bombarding BMS suspension-cultured cells and assaying for virus genome replication and protein expression.  Even more fortuitously, Janet Willment had been investigating the minimal cis-acting control regions for replication of MSV and their sequence specificity, and following Kenneth’s example, had set up exactly the right system for biolistic introduction of DNA constructs into suspension cultured cells as well as quantitative PCR for assay of replication, that Tich required for his work.  He was able to test three different constructs in conjunction with partial dimers of MSV-Kom by transient expression in biolistically transformed cells, and prove that they significantly inhibited MSV genome replication.  He got a great PhD, and has gone on to good things in the agricultural biotech sector in SA.

The stage was now set and dressed for the final act: this was the introduction of Dionne Miles, now Shepherd, into the continuation of this project into whole plant testing.  Here also was another example of fortuitous cross-bleed between projects: it happened that Kenneth Palmer had been helping Jennifer Thomson with a PhD student who was working on a difficult project to do with regeneration of cereals from anther culture.  He helped Wusi develop a system for transformation and regeneration of a model grass species, Digitaria sanguinalis, originally sourced from the flowerbed next to the UCT Sports Centre.  As it happened, some regenerated Digitaria was in a plant room that had escaped viruliferous leafhoppers in it – and got infected with MSV-Kom, and showed splendid streak symptoms as well as stunting.  As it was easier to transform than maize, could be grown as a perennial by simply cutting it back, and went to seed inside six weeks, it was obvious that we had a wonderful model plant for MSV resistance testing.  Another vital cog in what was becoming a complicated machine was my student Darrin Martin, who had developed a truly wonderful image processing-based quantitative symptom assessment tool as a central part of his PhD project on the determinants of pathogenicity in MSV.  This also proved vital in future work on accurate determination of the degree of resistance of regenerated maize.

download_springer_com_static_pdf_402_art_253A10_1023_252FA_253A1018659430217_pdf_auth66_1411045493_5ebfedf144f1424a1d7fa27c8f769370_ext__pdf

Dionne started what was to become her whole professional life to date in the late 1990s, by modifying Tich’s clones and testing them in maize cells for efficacy, and then introducing them into D sanguinalis cells for regeneration.  The value of the strategy was quickly apparent, when she showed that the best constructs for transient inhibition of MSV replication also either prevented regeneration of plants completely, or produced a very aberrant and infertile phenotype.

shepherd_MSV_resistance_pdf__page_6_of_12_

She was left with one – a truncated Rb- mutant Rep, rep1-219Rb- – that allowed regeneration of normal fertile plants and inhibited virus replication in transient assays, that has formed the basis of most of the work since. This includes the publication of her development of MSV-resistant transgenic fertile maize as “Maize streak virus-resistant transgenic maize: a first for Africain the Plant Biotechnology Journal in 2007, and getting us another cover.

Rybicki_biosci_june_2013_ppt

cover

What followed this was a long, painstaking grind by Dionne and team, and notably Marian Bezuidenhout who did most of the transformation and regeneration, in making as many transgenic lines as possible to provide to our maize seed producing industry partner Pannar Pty Ltd for introgression of the transgene into their elite breeding lines.  There followed much assessment of symptom development in greenhouse-tested plants, often grown from or descended from plantlets flown from our plant rooms to Greytown in an executive jet – along with boxes of wine, it does need to be said.  This has produced analyses that look like this: the product of more patient work than I think I would be capable of, and a really good example of how one should do this sort of work.  With repeats.  Many, many repeats.

Rybicki_biosci_june_2013_ppt

Dionne followed this up in 2011 with a second-generation product, with a paper on “A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay“:

“After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicative-form-specific PCR, it was clear that… the hairpin rep construct (pHPrepΔI(662)) completely inhibited MSV replication…[and] in addition, pHPrepΔI(662) inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity.”

This is also a big deal, as it represents another, alternative strategy to confer MSV resistance on maize, that confers wider resistance, and could potentially be stacked with the previous construct.

Then at last, we come to the present work – with a sense of the history behind it.  The rationale for this was the following:

“While we have previously developed MSV-resistant transgenic maize lines constitutively expressing “dominant negative mutant” versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or “leaky” expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture.”

Backtracking slightly, this has been the product of another networking experience at a foreign conference: this time, between me and my by-then-old friend James Dale from Queensland University of Technology, at the “Virologica 2001” conference of the Brazilian Society for Virology in Caldas Novas, Brazil.  Relaxing as invited speakers do, with a beer on a balcony, James said to me “Ed, mate, you’re going to kick yourself that you didn’t think of this!”, and proceeded to tell me about a geminivirus-based inducible expression system he and his group had just invented.  I did.  I also swore, loudly.  I then got him to offer highly reasonable terms for us to use the technology, and promptly took the idea home.  I have also described it in a recent review on ssDNA virus-derived plant expression vectors, so I take great glee in presenting my graphic here:

ssDNA_vector_Chapter_Rybicki__page_19_of_27_

This has only been published recently, due to patent issues and its use in proprietary production – however, we got the full cooperation of James and Ben Dugdale of QUT early on, and

“…used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.”

msv induc res

Pretty good, you’d think?  Also an excellent example of South-South collaboration, and investigations being set up as they should: by two people having a beer, at a conference.  Thanks, James!  Thanks Benno!  The paper ends optimistically, with:

“Ultimately, the practicality of the SGCs described in this study will only be fully realised with the regeneration of phenotypically normal transgenic maize plants engineered to contain the SGC that are resistant/immune to MSV infection. To this end we have regenerated a number of transgenic maize lines containing a SGC capable of expressing the most effective Rep mutant, namely RepIII-Rb-. In contrast to lines constitutively expressing this mutant gene, SGC lines have produced T2generation offspring with normal phenotypes.

Considering that only one strain – MSV-A – causes severe disease in maize throughout the whole geographical range of MSV, and that all isolates so far discovered within this strain have a maximum divergence of only 4.62% at the nucleotide level, it is likely that this novel MSV-inducible resistance construct will be effective against the complete spectrum of severe maize streak disease-causing African MSVs.”

Sadly, and these promises notwithstanding, this has all come to an end, with the non-renewal of the funding – possibly as a result of the takeover of Pannar by Pioneer HiBred Intl, but for whatever reason, it marks literally the end of an era.

What the hell – we had fun.  And maybe we can still sell it to someone.

Mucosal SIV Vaccines with Bacterial Adjuvants Prevent SIV Infection in Macaques

2 September, 2014

A new paradigm of mucosal vaccination against HIV infection has been investigated in the macaque model. A vaccine consisting of inactivated SIVmac239 particles together with a living bacterial adjuvant (either the Calmette & Guerin bacillus, lactobacillus plantarum or Lactobacillus rhamnosus) was administered to macaques via the vaginal or oral/intragastic route. In contrast to all established human and veterinary vaccines, these three vaccine regimens did not elicit SIV-specific antibodies nor cytotoxic T-lymphocytes but induced a previously unrecognized population of non-cytolytic MHCIb/E-restricted CD8+T regulatory cells that suppressed the activation of SIV positive CD4+ T-lymphocytes. SIV reverse transcription was thereby blocked in inactivated CD4+ T-cells; the initial burst of virus replication was prevented and the vaccinated macaques were protected from a challenge infection. Three to 14 months after intragastric immunization, 24 macaques were challenged intrarectally with a high dose of SIVmac239 or with the heterologous strain SIV B670 (both strains grown on macaques PBMC). Twenty-three of these animals were found to be protected for up to 48 months while all 24 control macaques became infected. This protective effect against SIV challenge together with the concomitant identification of a robust ex-vivo correlate of protection suggests a new approach for developing an HIV vaccine in humans. The induction of this new class of CD8+ T regulatory cells could also possibly be used therapeutically for suppressing HIV replication in infected patients and this novel tolerogenic vaccine paradigm may have potential applications for treating a wide range of immune disorders and is likely to may have profound implications across immunology generally.

 

Graphic of cells involved in HIV immunity from Russell Kightley Media

Source: journal.frontiersin.org

I have heard Jean-Marie Andrieu present this work – and I can understand why there is some skepticism surrounding it, because it is almost too good to be true.

Seriously: SUPPRESSING SIV-specific CD4 T-cell activation results in immunity to challenge infection??

However, and however – if this work is found to have been done well (and there is no evidence it was not), then this really could be a simple, reliable way of immunising people against HIV

Of course, monkeys aren’t people, and SIV is not HIV, so there MAY be a problem somewhere along the line in translating these results into humans – but what if there is not?

Then we may have a vaccine, and kudos to Jean-Marie Andrieu and co-workers to persevering along a difficult road to get their idea tested.

See on Scoop.itVirology News

20 years on, and here we are with Ebola, again

25 August, 2014

Browsing through my own web pages in an effort to clean up dead-end links, and cull tired material, I discovered that my link to an essay I wrote 19 years ago was still live – and as it referred to something written in and put up on our nascent Web server in 1994, means it has a 20-year anniversary round about now.

My essay is

The Student, the Web and the Ebola Connection

or:

Dr Jacobson, are you going to Kikwit?”

…and it is a record of events that resulted in 1994 from (a) an Honours student essay being written on “Emerging Viruses”, and (b) me playing around with the then-very-new WWW server that UCT has enabled – but didn’t tell anyone about, because they didn’t want anyone to use it until they had sorted out policies.  Oh, and (c) – the Kikwit Ebola outbreak in 1995.

I wrote in 1995:

“The whole phenomenon has been an object exercise in the power of the Web as a tool for the wide dissemination of information: we reached not only professional virologists, but also health-care professionals, and – most importantly – the lay public on a large scale”

And of course, this is even more true now – which is why, following the benign guidance of The Guru Cann, I maintain ViroBlogy and Virology News, and heartily recommend a Web presence to anyone who feels they need to disseminate information on topics of specialist and generalist interest to the world at large.

Of course, nearly all the links out of that essay are now dead – including to the original essay, that for a while there in 1995 was the ONLY detailed information on Ebola available on the Web.  So here is Alison Jacobson’s original essay, in full, revealed by going to my teaching material and checking out essays from 1997 and thereabouts:

EMERGING AND RE-EMERGING VIRUSES: AN ESSAY

Of course, I also maintained a daily update on the Kikwit outbreak, and then a couple of the next ones, before the Web caught up with me and it became easier to just trawl it for news via Google and its predecessors.  It still makes interesting reading, though, to go through some of what was posted from the disease frontlines back in the 1990s – and to remember that I had the TIME to do that kind of thing!

Where we are now

Well, here we are with what is the worst outbreak of Ebola in history, and here am I – again – trying to keep up with it.  This time, by the very excellent medium of the Web news aggregator Scoop.it, where I have established Virology News as a means of quickly and easily getting news out to the public.  Again, following the very excellent example of TGC, but also Chris Upton, who babied me along by letting me co-curate his Virology and Bioinformatics site.

Of course, there is a new angle to this outbreak – and that has been the compassionate use of a plant-made monoclonal antibody cocktail (ZMapp), hitherto only tested preclinically in a primate model.  Fortuitously, this all happened while I was finishing off a review on plant-made viral vaccines, so I reported on it – with references – here on ViroBlogy.

I was also able to report on it in my Plant Molecular Farming news site, with some authoritative statements from pioneers of the technology: Charles Arntzen from the Arizona Biodesign Institute sent through a link for an interview he did, and CNN covered it quite well too.  Charlie also sent through a set of links in an email that he was happy to share:

“The original story

There is a lot of interest from the press in “why tobacco” and “how does it work”?

The other focus is on the politics of scale up of the drug — it seems that criticism of the US is mounting in some sectors of Africa, and elsewhere.   I talked to a Spanish Language radio news station this morning, and the main questions related to “why is this a Secret Drug; are you trying to hide the secret from the world?”    “Is Reynolds tobacco trying to stop the supply of this drug to Africans?”    One guy asked if it was true that the Ebola Virus had been created in a test tube.

It seems that the press is largely to blame for using terms like Secret Drug.   It appears that they are also trying to mount political pressure to make a lot more of the drug to help Africans.   [This was] a nice job answering some of this….”

And at time of writing, the outbreak was still raging, had spread to Nigeria, and airlines were banning travel to half of West Africa – and alarmist tourist firms were advising people not to come to South and East Africa, as well.  The WHO has also said the impact is probably much greater than reported.

And Alison Jacobson is alive and well, and NOT working in virology any more.  Sadly!


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