Archive for the ‘biofarming’ Category

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!

5 Viruses That Are More Frightening Than Ebola

20 August, 2014

By Elizabeth Palermo, Staff Writer
Published: 08/15/2014 01:58 PM EDT on LiveScience
The Ebola virus has now killed more than 1,000 people in West Africa. Although the mortality rate of the most recent outbreak isn’t as high as in previous events, it’s still the case that most people who become infected with Ebola will not survive. (The mortality rate is about 60 percent for the current outbreak, compared with 90 percent in the past, according to the National Institutes of Health.)

1. Rabies

2. HIV

3. Influenza

4. Mosquito-borne viruses

5. Rotavirus

 

 

Source: www.huffingtonpost.com

Amen!  I have a fondness for Ebola simply because it is so spectacularly nasty, but it has killed fewer people in 40 years than flu or rotavirus does in 1.

Seriously: just like charismatic animals like elephants and tigers get all of the headlines when it comes to being endangered, rather than the humble tree frog(s), so do Ebola and Marburg get all of the attention when it comes to reportage on virus epidemics / pandemics.

See on Scoop.itVirology and Bioinformatics from Virology.ca

Plant-made antibodies used as therapy for Ebola in humans: post-exposure prophylaxis goes green!

5 August, 2014
Ebola virus budding from an infected cell.  Courtesy of Russell Kightley Media

Ebola virus budding from an infected cell.
Courtesy of Russell Kightley Media

Yes, I know you fans of ViroBlogy like Ebola – and just coincidentally, I was desperately trying to finish a review* on “Plant-based vaccines against viruses” against a backdrop of an out-of-control Ebola epidemic in West Africa, when three different people emailed me different links to news of use of a plant-made monoclonal antibody cocktail.  I immediately included it in my review – and I am publishing an excerpt here, for informations’ sake.  Enjoy!

Plantibodies against Ebola

The production of anti-Ebola virus antibodies has recently been explored in plants: this could yet become an important part of the arsenal to prevent disease in healthcare workers, given that at the time of writing an uncontrolled Ebola haemorrhagic fever outbreak was still raging in West Africa, and the use of experimental solutions was being suggested (Senthilingam, 2014). For example, use of a high-yielding geminivirus-based transient expression system in N benthamiana that is particularly suited to simultaneous expression of several proteins allowed expression of a MAb (6DB) known to protect animals from Ebola virus infection, at levels of 0.5 g/kg biomass (Chen et al., 2011). The same group also used the same vector system (described in detail here (Rybicki and Martin, 2014)) in lettuce to produce potentially therapeutic MAbs against both Ebola and West Nile viruses (Lai et al., 2012).

A more comprehensive investigation was reported recently, of both plant production of Mabs and post-exposure prophylaxis of Ebola virus infection in rhesus macaques (Olinger et al., 2012). Three Ebola-specific mouse-human chimaeric MAbs (h-13F6, c13C6, and c6D8; the latter two both neutralising) were produced in whole N benthamiana plants via agroinfilration of magnICON TMV-derived viral vectors. A mixture of the three MAbs – called MB-003 – given as a single dose of 16.7 mg/kg per Mab 1 hour post-infection followed by doses on days 4 and 8, protected 3 of 3 macaques from lethal challenge with 1 000 pfu of Ebola virus. The researchers subsequently showed significant protection with MB-003 treatment given 24 or 48 hours post-infection, with four of six monkeys testing surviving, compared to none in two controls. All surviving animals treated with MB-003 experienced insignificant if any viraemia, and negligible clinical symptoms compared to the control animals. A significant finding was that the plant-produced MAbs were three times as potent as the CHO cell-produced equivalents – a clear case of plant production leading to “biobetters”. A follow-up of this work investigated efficacy of treatment with MB-003 after confirmation of infection in rhesus macaques, “according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization” (Pettitt et al., 2013). In this experiment 43% of treated animals survived, whereas all controls tested here and previously with the same challenge protocol died from the infection.

In news from just prior to submission of this article, a report quoted as coming from the National Institute of Allergy and Infectious Diseases states that two US healthcare workers who contracted Ebola in Liberia were treated with a cocktail of anti-Ebola Mabs called ZMapp – described as a successor to MB-003 – developed by Mapp Pharmaceutical of San Diego, and manufactured by Kentucky BioProcessing (Langreth et al., 2014). Despite being given up to nine days post-infection in one case, it appears to have been effective (Wilson and Dellorto, 2014).

A novel application of the same technology was also used to produce an Ebola immune complex (EIC) in N benthamiana, consisting of the Ebola envelope glycoprotein GP1 fused to the C-terminus of the heavy chain of the humanised 6D8 MAb, which binds a linear epitope on GP1. Geminivirus vector-mediated co-expression of the GP1-HC fusion and the 6D8 light chain produced assembled immunoglobulin, which was purified by protein G affinity chromatography. The resultant molecules bound the complement factor C1q, indicating immune complex formation. Subcutaneous immunisation of mice with purified EIC elicited high level anti-GP1 antibody production, comparable to use of GP1 VLPs (Phoolcharoen et al., 2011). This is the first published account of an Ebola virus candidate vaccine to be produced in plants.

References

Chen, Q., He, J., Phoolcharoen, W., Mason, H.S., 2011. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human vaccines 7, 331-338.

Lai, H., He, J., Engle, M., Diamond, M.S., Chen, Q., 2012. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant biotechnology journal 10, 95-104.

Langreth, R., Chen, C., Nash, J., Lauerman, J., 2014. Ebola Drug Made From Tobacco Plant Saves U.S. Aid Workers. Bloomberg.com.

Olinger, G.G., Jr., Pettitt, J., Kim, D., Working, C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M., Whaley, K.J., Lear, C.M., Biggins, J.E., Scully, C., Hensley, L., Zeitlin, L., 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America 109, 18030-18035.

Pettitt, J., Zeitlin, L., Kim do, H., Working, C., Johnson, J.C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J., Pauly, M.H., Whaley, K.J., Ingram, M.F., Zovanyi, A., Heinrich, M., Piper, A., Zelko, J., Olinger, G.G., 2013. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science translational medicine 5, 199ra113.

Phoolcharoen, W., Bhoo, S.H., Lai, H., Ma, J., Arntzen, C.J., Chen, Q., Mason, H.S., 2011. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant biotechnology journal 9, 807-816.

Rybicki, E.P., Martin, D.P., 2014. Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants. Current topics in microbiology and immunology 375, 19-45.

Senthilingam, M., 2014. Ebola outbreak: Is it time to test experimental vaccines? CNN.

Wilson, J., Dellorto, D., 2014. 9 questions about this new Ebola drug. CNN.

* = which, despite their having commissioned from me, the good folk at “Viruses” an unnamed journal decided “…may not have substantial differences with the reviews you published recently” – and rejected.  I shall have revenge.  Oh, yes…B-)

Recombinant Bluetongue virus vaccines – or some, anyway

1 May, 2014
VIRUS-rota-200

General model of reo-like viruses. Copyright Russell Kightley Media

I picked up yesterday – via @MicrobeTweets’ Twitter feed – on a very useful list of papers in a “Virtual Special Issue” of Elsevier’s recent coverage of vaccines – for “World Immunization Week”. Great stuff, I thought to myself, as I browsed the list – and downloaded at least those that were Open Access, or which I can get via our Libraries’ IP range.

“Even better!”, I thought, as I saw a review entitled “Recombinant vaccines against bluetongue virus?”  A meaty, well-sourced review, I thought; good reading for me and my students / coworkers, and good meat for upcoming Introductions for papers yet to be written.  Indeed, it promised the following:

“The multiple outbreaks of BTV in Mediterranean Europe in the last two decades and the incursion of BTV-8 in Northern Europe in 2008 has re-stimulated the interest to develop improved vaccination strategies against BTV. In particular, safer, cross-reactive, more efficacious vaccines with differential diagnostic capability have been pursued by multiple BTV research groups and vaccine manufacturers. A wide variety of recombinant BTV vaccine prototypes have been investigated, ranging from baculovirus-expressed sub-unit vaccines to the use of live viral vectors. This article gives a brief overview of all these modern approaches to develop vaccines against BTV including some recent unpublished data.”

So, I parked the conveniently Open Access-ible window away on the side of my desktop, to be got back to with every expectation of delight.

Until I read it, that is: well-sourced it may be; excellent in its coverage, it is NOT.  In fact, apart from a brief discursion on subunit vaccines – concentrating almost exclusively on baculovirus / insect cell-produced proteins – it is almost exclusively concerned with live viral vectors for bluetongue proteins, and of poxviruses in particular.  Now, this is all very well, if that is what they work on – but to dismiss one of the potentially most exciting developments in recent Bluetongue vaccinology like this:

“VLPs of BTV have been also produced in plants recently using the cowpea mosaic virus and their use in a vaccination study produced no clinical manifestations in sheep after homologous challenge, although viremia was no [sic] evaluated (Thuenemann et al., 2013).”

- boggles the mind somewhat.  Really?  That’s all they have, compared to the screed immediately before it on baculovirus-produced antigens?  They get the expression system wrong – it is an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana involving a Cowpea mosaic virus-derived enhanced translation vector – and neglect to mention that the VLPs produced are as good as anything produced in insect cells; will be FAR cheaper to produce, and WORKED AS WELL AS THE CONVENTIONAL ATTENUATED LIVE VIRUS VACCINE IN A CHALLENGE EXPERIMENT IN SHEEP.  True!

This is a big deal, folks, really: successful production of significant amounts of VLPs requiring simultaneous expression of 4 structural proteins of BTV-8 in plants AND their subsequent assembly, AND performing as well as the standard vaccine in an animal trial.  But no – not good enough for our review’s authors….

I must declare vested interests up front here: first, we work on plant-made recombinant Bluetongue vaccines; second, I and others in my group are co-authors of the paper whose lack of coverage I am aggrieved about.

But that’s not the point: what IS the point is that this review is a slipshod piece of work that damns our collective endeavour with faint praise, in community that might otherwise have been alerted to an alternative to the far-too-expensive-for-animal-use baculovirus expression technology.

Ah, well.  I suppose that’s what blogs are for B-)

Eat your vaccines

7 February, 2014

See on Scoop.itVirology and Bioinformatics from Virology.ca

Vaccines have been revolutionary in medicine, but why are they not used in some parts of the world and how can they be improved? …

Ouch! Wouldn’t it be great if instead of a jab with a needle, you could just eat a vaccine instead? Luckily, researchers at the University of California agree, and their attempts to use algae to produce an edible malaria vaccine is just one example of the many strides forward scientists are taking in vaccine research.

Ed Rybicki‘s insight:

I love these idealistic but naive statements about how plant-production-of-vaccines-will-let us-get-away-from-needles: very 1990s; a little out of touch with modern realities – unfortunately!

The facts are that any edible (read: oral) vaccine will have to be regulated as tightly as an injectable, in terms of dose and administration.

Really: giving too little OR too much; giving it too often or not often enough; giving a product that has not been QCed or checked for potency  after storage…is suicide, in the vaccine world.

Even if it IS safe enough to eat.

See on www.isciencemag.co.uk

Legends of Virology

31 January, 2014

I have been fortunate enough this week to be in Pretoria, at the first Animal and Human Vaccine Development in South Africa Conference (Twitter #AHVDSA): partly because it is a very timeous and necessary meeting to help to establish strategies for this purpose, and partly because there is a significant presence of some legendary figures of international and South African virology.

Marc van Regenmortel – who we count as local even if he lives in Strasbourg – helped Bob Millar and others at the University of Pretoria to organise this meeting. He also used the opportunity of having a bunch of old virological friends visiting him at the University of Stellenbosch’s STIAS to bolster the conference presentations.

So it was that we have Errling Norrby of Sweden with us; we have Fred Murphy of Ebola fame; Marian Horzinek of veterinary virology repute; Marc himself, our iconoclastic viral immunologist; Jose Esparza of the BMG and an eminent poxvirologist – and Jean-Marie Andrieu, an oncologist with an interest in tolerogenic HIV vaccines.

Local legends are present too: we have Daan Verwoerd, legendary orbivirologist and former Director of the venerable and distinguished Onderstepoort Veterinary Institute; Henk Huismans, who did the first molecular work on orbiviruses in the 1970s, and is still active; Bob Swanepoel, doyen of the African haemorrhagic fever viruses.

Good people.

Oh, and of course, me and Anna-Lise Williamson; Dion du Plessis of OVI; Lynn Morris of the NICD; Albie van Dijk of UNW; Glenda Gray of the MRC, among 150 delegates

A great meeting, all in all, and very timely, given the contents of the SA Governmental Bioeconomy Strategy document released recently.

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Legends alive: from left, Fred Murphy; Daan Verwoerd; Bob Millar; Henk Huismans; Errling Norrby; Marc van Regenmortel
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Jean-Marie Andrieu; Marc van Regenmortel – at a VERY good unofficial dinner

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Legends and friends at supper: Marc, Fred, Eric Etter (CIRAD); Jose Esparza; Marian Horzinek; Errling, Anna-Lise Williamson

Emergency response vaccines for H5N1 influenza in South Africa

1 November, 2013

Our group has been working for some time now – since 2006, in fact – on investigating the feasibility of providing South (and southern) Africa with emergency response pandemic influenza vaccines.  The research was initiated after the Virology Africa 2005 conference that Anna-Lise Williamson and I organised in the Cape Town Waterfront in November of that year – when a senior WHO official warned us in his talk that “…if a pandemic hits, you are on your own: no-one will give you any vaccine”.

A group of us sat down afterwards, and discussed the feasibility of looking at emergency response vaccine(s), given that we had no capability in the whole of Africa to make flu vaccines.  Anna-Lise and I put together a proposal, with the highly pathogenic avian H5N1 influenza A as a target, which was funded on a once-off one-year basis by the Poliomyelitis Research Foundation (PRF) here in SA for 2006 – and then again by the PRF as a three-year Major Impact Project  (MIP) from 2008-2010, and subsequently to a lower level by both the PRF and the Medical Research Council of SA.  What made it all the more impressive for a South African project was that we had proposed expressing a protein-based vaccine in plants – quite a revolutionary prospect at the time, but something that followed on from the highly successful production of Human papillomavirus virus-like particles by transient expression in Nicotiana benthamiana by  James Maclean, working as a postdoc in our lab at the time.

However, some of the most important work was done early: James was very quick to get the haemagglutinin (HA) gene for the A/Vietnam/1194/2004 strain of H5N1 synthesised by GeneArt in Germany, and cloned into the same Agrobacterium tumefaciens plant expression vectors from Professor Rainer Fischer’s lab in Aachen, Germany, that had been used for HPV.  His initial work showed that large amounts of HA protein could be produced, both as soluble protein which lacked a membrane localisation domain, and as the membrane-bound form.  This work formed the basis for a patent application on the transient expression of H5 HA that has now been granted.

Subsequently, when the PRF MIP started, we employed Dr Elizabeth (Liezl) Mortimer and Ms Sandiswa Mbewana to further the work: with collaborators from the National Institute for Communicable Diseases (NICD) in Johannesburg and State Veterinary Services in Stellenbosch, this investigated transient and transgenic expression of soluble and membrane-bound forms and their immunogenicity, as well as a DNA vaccine consisting of the HA genes cloned into Tomas Hanke’s pTH vector.

The protein expression work was published in 2012, as well as being featured here in ViroBlogy at the time.

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What we had managed to show was that we could get excellent production of the H5 HA in both soluble and bound forms, and that especially the membrane-associated form of the protein was highly immunogenic, and elicited antibodies in experimental animals that were appropriately neutralising, indicating its suitability as a vaccine candidate.

Now this all happened despite our running out of money AND Liezl leaving to have a baby…and then we managed to get another paper out of the work, this time on the DNA vaccine side of things.

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We pitched this at the South African Journal of Science as a vindication of the faith in us by exclusively South African funding agencies – and managed to get the cover of the issue in which it appears, thanks to the truly excellent artwork of Russell Kightley from Canberra, Australia.  Front AND back covers, as it happens…!

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And this all made Sandiswa Mbewana, who is now a PhD student on another project, very happy:

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This all came in excellent time to mark the establishment in the Department of Molecular and Cell Biology at the University of Cape Town, of a new URC Research Unit: namely, the Biopharming Research Unit (BRU).

BRU

Watch this space…B-)


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