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.

Rabies Vaccine Protects Nonhuman Primates against Deadly Ebola Virus

26 October, 2014

The research team is pursuing the inactivated rabies/Ebola vaccine for use in humans. The live vaccine is being developed for use in protecting wildlife at risk of Ebola virus infection in Africa, which could also serve to prevent transmission into the human population.

Source: www.niaid.nih.gov

I missed this one at the time – and it is an interesting piece of news.  Basically, the research team cloned the Ebola envelope glycoprotein GP1 into the extant rabies virus vaccine strain genome, and tested a live version, a replication-deficient version, and a killed whole virion version in macaques.

Their results are interesting enough – 100% protection against challenge for live, 50% for the other two – that they plan to follow up to see whether or not additional doses could improve protection in the two non-replicating versions, and to make a “multivalent filovirus vaccine”.

This can only be welcome news against the backdrop of the still-ongoing epidemic in West Africa – where two other vaccines (recombinant vesicular stomatitis and chimpanzee adenovirus) are probably going to be trialled next year. The rabies version at least is based on a very well characterised vaccine that already protects against an extremely deadly disease – it remains to be seen how well the other two do.

I forgot to mention that I found reference to this article on “The Zombie Research Society”‘s blog site: http://zombieresearchsociety.com/archives/25562. A very apt place if one considers the parallels that are already being drawn between Ebola and a “zombie virus”.

And because I like zombies B-)

See on Scoop.itVirology News

Resurrecting Smallpox? Easier Than You Think

17 October, 2014

The virus’s genome is already online. You just need the right lab.

Source: www.nytimes.com

Weeeeeellll…yes and no. Smallpox is a BIIIIG genome – not far off in size to the bacterial genome famously resynthesised by Craig Venter et al., a while ago.  This means it would be a huge undertaking, cost a LOT of money, and need sophisticated facilities to do it.

Not something your average cave-dwelling fanatic could do, then!

States could do it, however: a well-funded lab in even a country like North Korea could theoretically resynthesise a poxvirus – but why bother??  We have vaccines against smallpox right now; growing poxviruses and vaccinia virus in particular is a well-established biotechnology still.

SO I think this is an artificial concern, to be honest. 

See on Scoop.itVirology News

Packs of wild dogs spread Ebola after eating corpses!! Or…not, maybe?

13 October, 2014

Packs of wild dogs spread Ebola after eating corpses

The ever-evolving Ebola narrative is broaching into ludicrous territory, with reports now claiming that wild dogs are going around digging up the rotting remains of deceased victims and eating their flesh in the streets. Special Ebola graveyards, where the dead are being buried in haste and at shallow depths, are reportedly feasting grounds for these dogs, which officials say are capable of spreading the disease to humans.

The Daily Mail says Liberian villagers first came across the dogs while going about their daily routines. Right in the middle of busy streets, they said, hungry hounds were allegedly seen ripping through rotting corpses, to the shock of onlookers. After determining the source of the bodies, it was revealed that shallow graves were to blame.

Source: www.naturalnews.com

Stephen Korsman of the Division of Medical Virology at UCT just alerted me to this article, in some distress because they had misquoted him and used his comments out of context.  This is a rather wild, sensationalist and highly inaccurate piece from a fringe web site that seems to have blocked me from commenting, because of previous criticism.  So, I’ll just do it here.

They comment: "Logically speaking, it makes little sense that asymptomatic dogs are possible Ebola carriers while asymptomatic humans are not. There exists no credible science to substantiate this apparent inconsistency beyond the baseless claims made by government health officials."

Utter garbage: bats carry Nipah virus, SARS-CoV, Ebola, Marburg AND rabies essentially asymptomatically – and can transmit ALL of them to other mammals. So too can deer mice transmit Sin Nombre hantavirus in the south-western USA without showing symptoms.  Rodents transmit Lassa fever virus in West Africa every year, again without being symptomatic.  Mice can transmit various South American haemorrhagic fever viruses without obviously being sick. I wish they would get their facts straight: this is is very easily checked!

See on Scoop.itVirology News

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

Effect of Formaldehyde Inactivation on Poliovirus

23 September, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

 

Source: jvi.asm.org

People have been treating poliovirus with formaldehyde for over 60 years – and it’s only NOW that someone thought to study in detail what happens!

I love this stuff: analytical centrifugation could have been done any time in the last fifty years (and has been, in determining structural transitions) but the newer techniques such as flow cytometry and RT-PCR could not. Analytically determining now what was empirically observed to work when polio vaccines were first made, is a historically important vindication of pioneering work that has almost made the viruses go away.

Simple and obvious findings, essentially – it is obvious that methylene bridging between amino acids would affect structural transitions; so too that HCHO treatment would kill viral ssRNA – but it hadn’t been DONE properly previously.  Great stuff!

See on Scoop.itVirology and Bioinformatics from Virology.ca

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.

Ebola: “We Could Have Stopped This”

8 September, 2014

Public health officials knew Ebola was coming. They know how to defeat it. But they’re blowing it anyway.

ld, you still just don’t get it. The Ebola epidemic that is raging across West Africa, killing more than half its victims, will not be conquered with principles of global solidarity and earnest appeals. It will not be stopped with dribbling funds, dozens of volunteer health workers, and barriers across national borders. And the current laboratory-confirmed tolls (3,944 cases, with 2,097 deaths) will soon rise exponentially.

To understand the scale of response the world must mount in order to stop Ebola’s march across Africa (and perhaps other continents), the world community needs to immediately consider the humanitarian efforts following the 2004 tsunami and its devastation of Aceh, Indonesia. The U.S. and Singaporean militaries launched their largest rescue missions in history: The United States alone put 12,600 military personnel to a rescue and recovery mission, including the deployment of nearly the entire Pacific fleet, 48 helicopters, and every Navy hospital ship in the region. The World Bank estimated that some $5 billion in direct aid was poured into the countries hard hit by the tsunami, and millions more were raised from private donors all over the world. And when the dust settled and reconstruction commenced, the affected countries still cried out for more.

Source: www.foreignpolicy.com

A seriously hard-hitting article by a very good journalist with a particular interest in infectious diseases.

And she’s right: Ebola was stopped, not once, but a number of times, as long as 38 years ago, in settings that are as or even more desperate in terms of poverty and lack of medics and medical resources.

The problem is, intervention did not occur soon enough this time, or on a scale sufficient to stem the increase in infections that inevitably followed introduction of the disease into urban settings.

It is a matter of amazement to me, that with the ever-present threat of pandemic influenza AND the recent emergence of MERS, that the WHO should have its "…miniscule epidemic-response department slashed to smithereens by three years of budget cuts".

Seriously: faced with diseases that can jump out of camels, or bats, or rats literally anywhere, WHO has to have budget cuts??

See on Scoop.itVirology News

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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