Archive for the ‘biotechnology’ Category

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-)

GMOs: poisons that will kill our children, or harmless foods?

29 October, 2013

I think I hung my colours out long ago in this “controversy”, but let us just be clear:

I DON’T BELIEVE ANY OF THE GMOs CURRENTLY BEING FARMED WORLDWIDE POSE ANY THREAT TO HUMANS OR STOCK ANIMALS AT ALL.  NONE!  NOR DO MOST BIOLOGICAL SCIENTISTS WHO ACTUALLY UNDERSTAND WHAT GENETIC MANIPULATION OF PLANTS ENTAILS.

Is that clear enough?  No ambiguity there?  Good!  Because the people who have taken poor Fair Lady magazine to task recently, mainly on their Facebook page, for daring to publish an article saying the same thing, would have you believe otherwise.  By relentless recycling of discredited animal feeding studies, reiteration of untruths, canards and plain lies, and by personal attacks on anyone expressing an alternate view.

Title page of the article

Title page of the article

I don’t think that Fair Lady will complain if I reproduce the title page, because I think their article is a reasoned, well written and factual exploration of the topic – which is a LOT more than I can say for most of the comments about the article.  Which includes gems like this:

“Oh dear Fairlady Magazine has made a BIG mistake!!!! Writing an article like this could put them out of business. I will never buy a Fairlady Magazine again and neither will any of my family. Stick to fashion Fairlady. Let Farmers Weekly publish an articles on GMO’s!!!! GMO’s are killing people. It’s not an exaggeration. It is proven, published, peer-reviewed fact. How many people do you know with cancer? Can you count on one hand or two. Ask yourself why. Maybe you could ask well-Informed People who are Fully Aware of the Irreversible Damage unleashed by Toxic GMOs on Earth.”

Now the problems that I have with the kinds of attacks on GMOs that are exemplified by these responses, are the following: these are the assertions that

  1. EVERYTHING is Monsanto’s fault
  2. ALL GMOs are toxic / poisonous
  3. There is ample evidence of harm to both animals and humans

All three of these straw men are, of course, rich in taurine excreta.  In the first place, while Monsanto may well have started the ball rolling on a big scale, and owns patents and seed rights on much of the early and simple one-trait GMOs, they do NOT own everything, and are NOT responsible for many of the recent developments still coming down the developmental pipeline – which are considerably more sophisticated than the ubiquitous herbicide-resistant or Bt-producing maize or cotton.  These would include plants resistant to various viruses, bacteria and fungi, plants engineered for higher nutrient / vitamin content (eg: Golden Rice and golden bananas), and drought- and salt-tolerant plants.

As for toxicity, NO GMO can be released if there is convincing evidence of toxicity in animal feeding trials, which HAVE to be conducted for each new “event”, or novel GMO.  I have sat on panels in SA which have assessed applications by seed companies to grow / produce GM crops, and I can tell you that this is a major feature of any application.  Where non-expert people often get confused is the fact that certain crop plants have been engineered to make insect-specific toxins normally produced by the bacterium Bacillus thuringiensis.  These are collectively known as “Bt toxins”, and the ones used as insecticides are specific for narrow ranges of related insects, and most often for lepidopterans – which include moths and butterflies.  Now the larvae of particularly certain species of moths are major agricultural pests, and include agents such as maize stalk borer and the cotton bollworm – and from Wikipedia:

“Spores and crystalline insecticidal proteins produced by B. thuringiensis have been used to control insect pests since the 1920s and are often applied as liquid sprays”.

That’s right: crystalline protein masses extracted from live bacteria and live spores of bacteria used to be sprayed around as pest-control agents.  Everywhere!  Moreover, from Wikipedia again,

“Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humanswildlifepollinators, and most other beneficial insects and are used in Organic farming“.

Yes, really: actual Bt toxin, and actual spores that can develop into live bacteria, can be used in organic farming.  Now why would anyone have a problem with a technology that LIMITS exposure of the environment to a bacterial toxin, and most especially, to live bacteria, by containing the protein within the plant tissues?  Moreover, the amount of Bt in the edible seeds of maize is minimal, and people don’t eat cotton – so we are left with possible effects on wildlife, and cattle which eat the green parts of the plants.  And no-one has ever  shown any deleterious effects of Bt in GM plants on non-target organisms.  Oh, there was the Pusztai report, which claimed to have shown that snowdrop lectin-containing transgenic potatoes were toxic to mice – but this elicited the following comment:

“The [British] government’s Advisory Committee on Novel Foods and Processes(ACNFP) has dismissed Dr Pusztai’s findings as inconclusive and irrelevant due to serious doubts concerning the design of the study. The particular type of potatoes on which Dr Pusztai conducted his experiments would never have been approved for food use. Indeed, the ACNFP stated that had an application been submitted on the basis of the data collated from this flawed study, it would have undoubtedly been rejected”

A nice exploration of the pervasive effects of bad publicity following publication of bad science was published recently: this was “When bad science makes good headlines: Bt maize and regulatory bans“, in Nature Biotechnology.  These authors state that

“Numerous laboratory toxicity studies and field experiments, as well as years of field observations in countries where Bt maize is cultivated, have provided evidence that the Cry1Ab protein expressed in Bt maize does not cause adverse effects on arthropods outside the order Lepidoptera (butterflies and moths), the group that contains the target pests. Supporting data have been analyzed in reviews and meta-analyses”

Another point of contention is herbicide-resistant plants, which again, have not convincingly been shown to be toxic.  I say convincingly, because anti-GMO activist will immediately quote “the Seralini study” which purportedly showed deleterious effects on lab rats fed transgenic maize producing a protein which detoxifies the herbicide glyphosate as well as the herbicide itself – to which I reply by inviting you to read this rather damning report by the European Food Safety Authority, which opens with the following statement:

“Serious defects in the design and methodology of a paper by Séralini et al. mean it does not meet acceptable scientific standards and there is no need to re-examine previous safety evaluations of genetically modified maize NK603″

Now I will remind everyone that this is an agency which is NOT in Monsanto’s pocket – or anyone else’s – and which upholds high standards in safeguarding the general public.  As do the US Department of Agriculture (USDA) and the Food & Drug Administration of the USA, which also have no problems with GMOs (FDA statement; USDA information).  Here is a another comprehensive refutation of the “evidence of toxicity” of glyphosate-resistant soybeans, an unpublished study that is widely quoted by anti-GM lobby.

As for “ample evidence of harm” – I can only refer you to what we biotechnologists would regard as an authoritative source, which is the journal Nature Biotechnology.  In a recent article on GMOs entitled “How safe does transgenic food need to be?” by Laura DeFrancesco, the author asks the question:

“Why, after transgenic products have been in the human food chain for more than a decade without overt ill effects, do these doubts persist? And will it ever be possible to gather sufficient evidence to ameliorate the concerns of skeptics and the public at large that these products are as safe as any other foodstuff?”

Further on, she says:

“Critics and proponents of genetically modified organisms (GMOs) alike agree that genetically modified foods have failed to produce any untoward health effects, and that the risk to human health from foods contaminated with pathogens is far greater than from GMOs” [my emphasis]

I don’t think I need to belabour the point further: I am hopelessly compromised, in the eyes of some of the more rabid activists, by being a biotechnologist at all, and especially – Gasp!! – BECAUSE MY LAB MAKES GMOs!!!  However, if that makes me more amenable to believe actual evidence-based findings, rather than unsubstantiated media hype, then so be it.

DNA Preparation Tubes Contaminated with Novel ssDNA Virus

21 September, 2013

See on Scoop.itVirology News

A novel virus thought to have come from human samples appears to have been derived from seawater during the manufacture of tubes used to extract DNA.  

Ed Rybicki‘s insight:

I have a problem with the original report, in J Virol (http://jvi.asm.org/content/early/2013/09/05/JVI.02323-13.abstract?related-urls=yes&legid=jvi;JVI.02323-13v1): not that they discovered it, because that was done well.  However, they essentially REdiscovered something that was ±100% identical to a virus already sequenced and named by Chinese researchers – who did not use the Qiagen kits, apparently – and then gave it a new name!

 

Sorry, that is simply bad practice!  It also smacks of scientific imperialism of a sort that characterised early discovery work on HTLVs and on HIV, when US researchers calmly treated earlier characterisations as if they had never happened.

 

There is another leap that I do not think is justified: the authors claim that

 

"Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture".

Really?  On the basis of presence of a sequence in a metagenomic trawl?  No resampling with specific primers on a fresh sample?   And surely the generation of the silica matrix is done under conditions that would totally destroy adventitiious DNA?

So – an interesting paper, and a valuable notification (although it might have been nicer if they’d shared their findings informally, to save other people like our Virology Diagnostics lab time and money).  But flawed, in my opinion.

See on www.the-scientist.com

Plant-Based Antibodies, Vaccines and Biologics 5, Part 5

3 September, 2013

Session 6:Vaccines II

This was SUPPOSED to open with a report from Medicago Inc, on ‘Developing plant-made influenza vaccines: From discovery to commercial scale production’  - but didn’t, because they were all shaken up (in a good way) by having been effectively bought by Mitsubishi Tanabe Pharma Corporation, and no-one came.

This is a success story in its own right, however, as their recent and highly successful activities in the areas of making influenza vaccines and human rotavirus VLP-based vaccines in plants marked them out as a target for acquisition by Big(gish) Pharma – for which we commend them.

It is sad, however, that their only presence at the conference was on the back of my windbreaker B-)

Konstantin Musiychuk (Fraunhofer USA) was the first up, then, speaking on ‘Preclinical evaluation of VLP-based malaria transmission blocking vaccine’.  He described how there are 3 types of intervention that may work with malaria: these are at the pre-erythrocytic, blood stage, and transmission blocking stages of infection.  Antibodies to Pfs48/45, Pfs230 proteins block the fertility of or destroy the macrogamete.  Pfs25 and 28 Abs block the ookinete to oocyst developmental phase; all potentially block transmission.  Accordingly, they expressed these as fusions with the alfalfa mosaic virus (AMV) CP with mutation(s) to prevent glycosylation.  The Pfs25 protein was the best candidate; they cloned a mutated version (glyc-), fused at the N-terminus to AMV CP, and expressed via their TMV-based “launch vector” after vacuum infiltration.  He noted that the fusions have full-length and proteolysed products – which is needed for VLP formation as native CP is needed to avoid steric hindrance in assembly.  They obtained nice particles as shown by EM, showing surface decoration.  Dynamic light scattering [Ed: must get me one of those…] results show a nice tight range of 17nm particles.

They used the products with/out Alhydrogel as adjuvant, IM in mice: they got good titres maintained >170 days, with  2x inoculation.  They diluted test sera with naive human serum and used this to membrane-feed mosquitoes, then after 1 week dissected them and assayed for parasites: oocyte counts in mid-gut reflected efficient blocking of acquisition.  The adjuvant+ doses worked well down to 0.1 ug (100%).  Single doses of 1, 5 or 25 worked 100% as well.  After 6 months, 5 and 25 ug doses still gave 90%+ blocking.

They made GMP lots, very pure:  2 doses at 0 and 21 days resulted in complete blocking down to 0.3 ug, with >99% blocking after 40+ days.  Tox studies were fine, although the  Alhydrogel apparently causes some side effects.   Scaleup from 1-50 kg showed no changes in the Ag.  The Phase 1 trial is expected in Q3 2013.

This was most impressive: it is to be hoped that the promise is maintained!

Yoseph Shaaltiel (Protalix Biotherapeutics, Israel) spoke on Protalix’s new product: this was alpha galactosidase-A, for the treatment of Fabry disease.  This is an X-linked lysosomal storage disease that results in massive storage of glycolipid Gb3, in cells, in the vascular system and elswhere, which impairs the tissue of the heart and affects kidney and other organ function.  There were worse consequences than with Gaucher disease, while it was less obvious.  The current therapy was seen as being bad, and patients had reduced life expectancy.  There were 2 therapeutic enzymes on the market: these were Agalsidase Alfa and Beta; these were very inefficient and expensive, so cost benefit was very limited.  1/2 life in blood was normally just a few minutes, and the proteins were very immunogenic.

Protalix aimed at making a biobetter: this was made in tobacco cells cultured in bags (they used Icon vectors, so could not work in their favoured carrot), by cocultivation with Agrobacterium and then killing the bacteria.  The protein subunits were PEGylated to reduce immunogenicity and x-linked using bis-NHS-PEG.  This gave improved stability, longer circulatory 1/2 life, enhanced activity in target organs with similar to improved kinetics, so lower dosing and longer intervals between doses were possible.  Yields were good too, and they could make the enzyme very pure.  The product had the same kinetics as the commercial products with better activity over a wide pH range.

As far as glycosylation was concerned, the commercial product had very complex glycosylation, while the plant-made product’s profile was very consistent and simple.  It had an enhanced circulatory 1/2 life, of 581 vs 13 min, and also had higher activity in target cells – heart, kidney – over time.  Yoseph noted that the  patents on the enzyme(s) were limited to CHO cell production, meaning they had a useful window to exploit.

A comment from Jim Carrick was that the FDA was not interested in PEGylated products, as this could lead to vacuolation of kidneys in the long term.  Yoseph said their product was not the same, as normally PEGylation added 20-40 kDa, whereas theirs was a much shorter x-linker.  Their product was, moreover, already in clinics, as the  FDA had said they should move straight to patients rather than testing it in healthy people.

Lydia Meador (Arizona State University) reported on their lab’s HIV vaccine candidate, made in plants and also vectored by NYVAC-KC delB19 poxvirus.  They had previously shown that a CTB-HIV membrane proximal region (MPR) fusion vaccine resulted in Ab that stops transcytosis of HIV by Ab; she noted that live vectors enhance T-cell responses compared to subunit vaccines, so a combination would be a good idea.

Accordingly, they had cleverly produced whole HIV Gag and a deconstructed gp41 – stable Gag transgenics, and transiently-produced dgp41 – in the same plants, to make 100nm VLPs.  While VLPs are highly immunogenic alone, they wanted to prime with the NYVAC and boost with plant-made antigen.  They obtained good p24 Ab responses with NYVAC and the VLP boost; gp41 less so.  In terms of mucosal immunity, they saw the IgA response against gp41 was significantly higher in the NYVAC+VLP combination, as were CD8+ T-cells.  She noted that the anti-NYVAC titre was high after 3x doses.  In response to my question, she did not know if the NYVAC vaccine made VLPs in mice – which it may not do, even if it works in plants, due to different protein requirements for budding in mouse vs plant cells.

Daniel Tusé (Intrucept Biomedicine, Kentucky) – a company founded with Kenneth Palmer – spoke on ‘Safety and efficacy of plant-produced Griffithsin for antiviral indications’.  He noted that while griffithsin was an excellent anti-HIV microbicide, it was also a reasonably broad-spectrum antiviral lectin, as it was effective against the recently-emerged MERS CoV and  influenza viruses.

The protein was hard to make from seaweed, and E coli was useless for production; however, they got g/kg in tobacco via conventional rTMV vectors, and now even better with Icon and Nomad vectors.  KBP had manufactured it to near-GMP production standards, again at g/kg yields, with product recovery at 30% from leaves and 50% from leaves + stems, to a final purity of 99.8%.  The potency was the same as the alga-derived product, and they had 100s of gm of product.

As griffithsin binds HIV with very high affinity, its primary use would be as a topical microbicide, to prevent transmission of HIV and HSV; to prevent coronavirus infections, and to act on chronic virus infections.  The protein is not mitogenic on PBMC and does not activate T cells; it does not produce inflammatory cytokines in human PBMC, unlike cyanovirin, which had a much worse proinflammatory profile.  The epithelial toxicity was also very low, which was in contrast to some well-publicised agents which had disastrously resulted in increases of HIV acquisition in women using them.

A carbopol-based gel was found to have the best drug-release kinetics, so was adopted for formulating the product for use.  This protects mice against genital herpes: herpes has 2x the risk of infection per exposure compared to HIV infection.  The gel has broad specific activity against coronaviruses too, to a wide spectrum of viruses from human, cow, chicken and pig.  It could protect mice against SARS CoV, if given intranasally at 2 doses/day.

The protein also has uses in prevention of infection in the organ transplant area, eg against hepatitis C virus (HCV): it prevents infection of Huh-7 cells by cell-culture derived HCV, and partially protects hepatocytes from viral spread in vivo.  If injected in animals it persists, and maintains an anti-HIV activity.  It is immunogenic, but only weakly so, and Ab to it don’t neutralize its effects.  Their lab was using rational design to take out T-cell epitopes without affecting antiviral activity.

Daniel stressed that this is a new drug, which can be preferentially be made in plants at high yield, with very low cost of goods; that it was effective and safe.

Hugh Haydon (KBP) mentioned that the cost of goods was “pennies/dose”.

Session 8:

This was an interactive discussion session, addressing the topic ‘Commercialisation of molecular pharming products – objectives and targets for the next 5 years’.

The panel: from left - Hugh Haydon, Kevin Whaley, John Butler, Scott Deeter, Einat Brill

The panel: from left – Hugh Haydon, Kevin Whaley, John Butler, Scott Deeter, Einat Brill

Hugh Haydon of Kentucky BioProcessing (KBP), , speaking on behalf of the new MAPP, KBP and Icon collaboration, addressed product selection.  He noted that MAPP was responsible for product development, Icon for technology development and purification, and KBP for large-scale manufacture.  They had spun out Solmab as a collaborative vehicle for production of MAbs for infectious disease therapy.

He described their product selection rationale: this was based on

  • proof of concept data
  • platform suitability
  • capacity for dual use of product
  • availability of capital
  • speed of the regulatory process
  • regulatory success rate
  • scalability of existing infrastructure

Accordingly, they had selected a “biobetter” of Synagis, and an Ebola MAb cocktail.  The Synagis equivalent was better due platform parameters, known clinical parameters, the fact there were established markets which can grow, government and NGO humanitarian interest, and potential adaptation to other viruses.  For Ebola, they had a 3 MAb cocktail that was known to work, strong government interest (for a stockpile), a more rapid regulatory pathway, and a tropical disease voucher from the FDA.  He pointed out that these products won’t make blockbuster status, but are appropriate for small companies like theirs.

Kevin Whaley (MAPP) spoke on how we needed therapeutics that were multipurpose (disease, indication) as well as multi-vaccines.  The attributes of the new biologics were multi-use, speed of production, scale of production, and cost advantage – especially for global health products costing <$US10/g, at scales of >10K kgs, with increased efficacy (pathology, cancer), increased acceptability and access.  He noted that all modern paediatric vaccines are multi – this saves visits to clinics, especially in developing countries.

Scott Deeter (InVitria) noted that the biologics market was edging up to being worth $US125 billion – and reckons progress with plant-produced products is excellent.

John Butler (Bayer) thinks we are still looking for suitable products!  He was of the opinion that initial targets were too difficult (eg NHL – and flu??!), and that improved product characteristics must benefit from being plant-made.  He was adamant that PMP must not compete on price with other platforms – because there was no such thing as a bottleneck in fermentation capacity world-wide, and established industry could just cut prices if they wanted to.  He spoke of real and perceived hurdles:

  • regulatory pathway isn’t a hurdle
  • plant vs human glycosylation is not either, as plant-specific glycans were not more immunogenic than human

Real risks were that:

  • there were well-established alternatives
  • the plant-made product industry was overstretched in terms of resources

Einat Brill (Protalix) addressed their future strategy:

  • new biologics for orphan indications (clinical trials were smaller, one needed only several 10s kg a year for an entire disease cohort)
  • recombinant vaccines
  • hard to express proteins that were best expressed in plants

ApApproved biologics:

  • Biobetters of commercial products
  • They would continue to establish PMP regulatory environment as a viable route for biologic drugs development
  • Biobetter efficacy: longer circulatory half life for favourable clinical outcome
  • regimen frequency: longer treatment intervals due to increased drug stability, with lower dosing
  • Changing administration route (eg: oral vs injectable): helps to improve patient compliance

This was an excellent session, if only to hear how people who have been involved in getting PMPs to the market viewed the prospects for the industry – and it appeared favourable, despite John Butler’s caveats.

Plant-Based Vaccines, Antibodies and Biologics 5, Part 4

2 September, 2013

PBVAB 5 Part 4

Sessions 5 – 8

The fifth session on Day 2 was “Antibodies 1” – and who better to kick off, than Rainer Fischer (RWTH / Fraunhofer Institute, Aachen), talking about Pharma-Planta – The European project to introduce plant-derived monoclonal antibodies to the clinic’.

One of the most impressive features of the FP6 PharmaPlanta project was its sheer size: 28 academic institutions were involved over 7 years, at a cost of €12 million plus €3 million from the Fraunhofer Institute in Aachen.  Their mission was to move molecular farming beyond proofs of concept, and to develop candidate products.  They selected the anti-HIV-1 subtype B MAb 2G12 as their final candidate, but also developed MAbs to rabies and some vaccine candidates.  Importantly, their IP had a Humanitarian Use Commitment: knowledge created was made freely available for humanitarian purposes.

They had a total of 39 postdocs and 8 students trained; they produced 200 peer-reviewed publications consisting of 150 research papers and 50 reviews, and a spin-out company.  The project also helped to develop a South African plant-made MAb production platform.  Their plant-produced 2G12 was the first plant-made MAb in human clinical trials – and went from gene to clinic in just 7 years.  They had also very materially helped the development of the regulatory regime in Europe, from the viewpoint of pharmaceutical guidelines and environmental safety for PMPs.

Rainer Fischer

Rainer Fischer in full flow

The final yield figures for 2G12 were 5 g of 97% pure MAb from 240 kg of transgenic tobacco, with a recovery of 55%.  The product had a better glycosylation homogeneity than CHO cell-produced 2G12.  In clinical trials of the MAb used as a vaginal microbicide, the product was safe and well tolerated with no serious adverse reactions.  There were no anti-Abs found in serum or in the vagina, with no systemic absorption.  The MAb survived for 8 hrs in the vagina, meaning it had serious potential as a microcode.

The project resulted in great human capital, a manufacturing facility at the Fraunhofer IME, and a number of important follow-on projects.  It also opened bottlenecks in regulatory practice, and in clinical trials of PMPs.  There was a pipeline of additional product candidates, eg anti-rabies MAbs.

Important lessons from the project were the following: one should focus early on on the plants used, the expression technology, the threshold level of production, realistic timelines, the plant line and purification process, production issues, QC stability, regulatory contract – FIND A CLINICAL SPONSOR!, set up contractual framework, draft specifications for drugs, contact authorities in countries for manufacture and testing.

Issues such as smart product selection, synthetic biology/host cell line engineering, glycan/protease profile, hi-throughput cloning, selection of elite lines, scale-up automation / vertical farming, downstream processing, regulatory approval had also surfaced, and were important.

For the future, a fully automated vertical farm unit  for seed development was going to come on stream.  They would move from niche production to mainstream production, taking advantage of economies of scale.  Other developments could be designing an optimal host cell line, with fully human glycosylation, and site-directed transgene integration.

Some day someone should write a book about this endeavour – and I think it should be Rainer.

Larry Zeitlin (MAPP Biopharmaceutical) spoke next, on producing monoclonals against respiratory syncytial virus (RSV): the reason for doing this is that RSV is a major pathogen among small children worldwide, and while there are MAb-based therapeutics (eg: Synagis, from MedImmune), with sales in the order of USD 1 billion annually, these cost around USD 5 000 for one treatment for one child – and premature infants or cardiac / respiratorily challenged children required 4-5 monthly doses per RSV season.  Additionally, infection with RSV in the 1st year of life is associated with development of asthma later, so paediatricians were wanting to treat a much wider spectrum of children.

Accordingly, MAPP was making a Synagis equivalent via Icon vectors in N benthamiana for half the cost of goods, which had the same neutralisation ability and same affinity but a different glycosylation profile and shorter half-life.  When tested in cotton rats it was identical in pharmacokinetics and worked as well as Synagis.  An attempt to reduce the interaction of the IgG1-based MAb with the immune system by changing the subtype to IgG2 failed in rates even though it was neutralising, possibly due to there being less ADCC.  Larry mentioned that they could engineer the Fc region with point mutations to significantly extend the half life – and then use this as a scaffold, possibly for some of their other products.

Michael McLean (Univ Guelph, Canada) described his group’s work on a HIV Ab cocktail theoretically capable of neutralising 99% of HIV strains – this was for PlantForm Corp, who had a mandate to produce biosimilars and novel biologics using plants.  The HIV project was focused presently on demonstrating anti-HIV functionality, and at improving glycosylation profiles of a cocktail of b12, 2F5 and 4E10 broad-spectrum anti-HIV MAbs.

They worked with BeYDV-derived, 2-replicon vectors expressing whole MAbs, as well as their own vectors, using the Steinkellner group glycosylation pathway engineered plants.  With 9 days maximum expression period  they could get 1 g/kg maximum yields.  All the MAbs worked fine, with  similar activity in in vitro HIV pseudovirion neutralisation assays.  Using the deltaFX N benth line, they get uniform glycosylation – and add Gal using their own vectors.

Shawn Chen (BioDesign Inst, Arizona State Univ) described their work on a humanized West Nile virus (WNV) therapeutic MAb which protected mice from WNV infection.  They wanted blood-brain barrier (BBB)-permeable bifunctional Abs to extend efficacy, presently limited because of the barrier.  They got 0.3 – 0.5 g/kg yield of a bifunctional MAb which bound the BBB endothelial receptor and virus Ag, using Icon and BeYDV vectors, and showed endocytosis into brain cells.  He also mentioned that they could “tune” glycoforms to change ADCC.

IMG_0140

Victor Klimyuk (Icon Genetics GmbH, Germany) presented on ‘Biogeneric antibodies made in plants’: these used a generic IgG1 constant region gene codon-optimised for plants, with add-on variable (V) regions derived from other Abs of different types and specificities.  The first product had been the non-Hodgkin lymphoma personalized MAbs: they had done glycotyping of each NHL MAb, all with the same H but diff L chains, to show these were differently glycosylated – and that all the idiotypes were expressed at very different levels.  Interestingly, expression levels had little to do with occupancy of glycosylation sites – and this occupancy could be tuned by directed point mutations.

They had made analogues of trastuzumab and herceptin, etc – and noted that herceptin analogues differed in potency, and wt plants produced lower levels than their engineered plants.  Rituximab analogues were all the same as the original MAb at day 0 of treatment, but MAbs with no fucose were best at persistence – equal to the original.

Vikram Virdi (VIB, University of Gent, Belgium) described passive immunisation of piglets against enterotoxigenic E coli (ETEC) using llama-derived antibodies produced in Arabidopsis.  This was useful in that it extended the maternally-derived passive immunity.  Their product was a “porcinised camellid Ab” against the major adhesion molecule of ETEC, which should survive the digestive tract.  They made MAbs based on a camellid Vh gene fused to IgG and IgA Fc regions, and expressed them in seeds for a piglet feed challenge.  They got a maximum of 15% TSP expressed in seed, 3% of seed weight.  By triple transformation with the 3 genes required for an IgA analogue (Vh:Fc, J chain and secretory component) and then selfing and breeding plant lines, they got in planta assembly of a sIgA analogue (0.2% seed weight).  This worked in inhibiting attachment of  bacteria, so they upscaled production and tested a cocktail of IgG vs IgA types.  The latter was best, with a swift decline of bacterial shedding with a 4  x lower dose than for IgG.  There was also a better weight gain for IgA treated piglets.

Thomas de Meyer (VIB-PSB/University of Gent) compared production of bivalent camellid VHH-derived MAbs in Arabidopsis, N benthamiana and Pichia pastoris, given that the VHH Fc enhanced functional affinity, and led to longer serum 1/2 life, and was a convenient protein tag. They compared VHH and VHH-Fc MAbs with 4 fusions, including anti-globulin, anti-albumin, and anti-GFP.  The products were stable in seed production (with KDEL) in Arabidopsis and also N benthamiana, and  Pichia secreted the products.  They got yields of 1.5 – 27% TSP, 0.1 to 0.82 g/kg in plants, and with Pichia, 15 – 30 mg/l culture.

The MAbs had different size profiles from the different hosts, though all were bivalent VHH, and N benthamiana and Pichia products were fully glycosylated.  Several of the Fc-type MAbs outperformed the VHHs in ELISA.

Overall, it was obvious that expression of a wide variety of antibodies in plants is a maturing technology: yields are high, of antibodies whose glycosylation and retention profiles can be handily engineered, and which perorm equivalently or better than their conventional homologues in in vitro and in vivo assays.

Go Green, he said, not quietly…B-)

PBVAB 5 – Part 3

21 August, 2013

PBVAB 5 Verona, June 2013 – Part 3

Technically, Sue Huddy’s piece should have been Part 3; however, it reports things that happened after what I am reporting on, so I’ll keep that label!

This post will report on Sessions 3 & 4, namely, Technology Advances and Perspectives.

I opened Session 3 with a talk on ‘Virus-derived ssDNA vectors for the expression of foreign proteins in plants’, focusing mainly on geminiviruses (naturally).  I wrote this a couple of years ago as a chapter for a book which seemed to not be forthcoming; however, I was assured during my talk by Yuri Gleba – the co-Editor with Kenneth Palmer of a “Current Topics in Microbiology and Immunology” issue on “Plant Viral Vectors” – that this offering is now in fact available, so here’s a link for anyone who wants to buy it.

Current Topics in Microbiology and Immunology 2011,

Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants

Edward P. RybickiDarrin P. Martin

Plant viruses with ssRNA genomes provide a unique opportunity for generating expression vehicles for biopharming in plants, as constructs containing only the replication origin, with the replication-associated protein (Rep) gene provided in cis or in trans, can be replicationally amplified in vivo by several orders of magnitude, with significant accompanying increases in transcription and expression of gene(s) of interest. Appropriate replicating vectors or replicons may be derived from several different generic geminiviruses (family Geminiviridae) or nanoviruses (family Nanoviridae), for potential expression of a wide range of single or even multiple products in a wide range of plant families. The use of vacuum or other infiltration of whole plants by Agrobacterium tumefaciens suspensions has allowed the development of a set of expression vectors that rival the deconstructed RNA virus vectors in their yield and application, with some potential advantages over the latter that still need to be explored. Several modern applications of ssDNA plant vectors and their future potential will be discussed.

I noted that several firms are already using geminivirus-derived expression technology – like Kentucky Bioprocessing, who offer use of it as a service, and Medicago Inc, who use it in manufacturing vaccine products – and that it has considerable potential for improvement.  There is also the possibility of using other ssDNA virus-derived vectors, including from bacteria.

E.V. Sheshukova (N.I. Vavilov Institute of General Genetics RAS, Moscow) followed up with an account of how the use of antisense RNA to plant death factor (PDF) could modulate PDF level so as to avoid the necrotisation caused by rapid protein over-expression.  Their group used a TMV-based vector to co-express an antisense with the gene of interest, and got 4-5-fold increase in protein expression, equivalent to using the silencing suppressor p19 from a tombusvirus.

Diego Orzaez (IPMCP-CSIC, Valencia, Spain) spoke next, on the same technology I have previously described (with beautiful pictures from Diego) here: that is, the enabling of tools for multigene engineering of plants – and specifically in this case, the elegant use of superinfection exclusion phenomenon seen with RNA plant virus-derived vectors that are capable of movement, for the expression of polyclonal antibody mixtures in plant leaves.  They had successfully shown expression of 300+ individual clones from a camel VHH clonal library derived against a mixture of 3 snake venoms, in a mosaic on a single leaf.  This was seriously impressive for me: imagine, polyclonal “sera” from a leaf!

Diego noted that the FDA allows the 2-animal rule for products like antivenin, and things used for biodefence: that is, an efficacy trial in an animal, followed by Phase 1 trial in humans (=safety).  This could help expedite approval of such products.

We discussed the paper previously blogged on from this group in Journal Club today, incidentally, to much appreciation of the truly excellent work, and the colour Figures.  Thanks, Richard!

Reza Saberianfar (Agriculture and Agri-Food Canada, Ontario) described their investigations of protein body biogenesis in N benthamiana.  They had looked mainly at hydrophobin and elastin fusion proteins, in order to overcome the joint bottlenecks of inadequate accumulation, and difficulties in purification of recombinant proteins from plants.  He noted that hydrophobin and elastin PBs were different sizes: they had used protoplasts of infiltrated leaves and confococal microscopy and Imaris software to find every PB in individual cells, to determine that  shows hydrophobin-based PBs were 1-2 um, and ELP-based were 2-3 um in diameter, for the same amount of protein.  PBs made from  hydrophobin and ELP-linked proteins shared the same ER origin, but Zera-based PBs had a different origin and Zera fusions did not need a KDEL for ER retention.  An interesting observation was that PBs could form in the ER in the absence of fusion tags if expression levels were high.  One could also increase the expression of other proteins by coexpressing them with a fusion protein, as they get incorporated into PBs anyway – eg: EPO.

Lauri Reuter (VTT-Technical Research Centre, Finland) continued in the theme of fusion proteins with a talk on the production of hydrophobin fusions in tobacco BY-2 suspension cultured cells.  It was interesting to hear that WAVE bioreactors did not work well because they did not shake fast enough, but that conventional steel bioreactors did – with capacities of 20 – 600 litre, and even up to 20 m3.  The cells are apparently surprisingly tolerant to shear stresses, and yields of GFP::hydrophobin fusion from 600 litre reactors were as good or better as from a 50 ml shake flask – at 300 mg/litre.  Purification was simple, in that reactors could be pumped out onto a filter, and the cell “cake” pressed dry – for subsequent lyophilisation and storage at room temperature, for example.  French pressing of fresh cells was also an option.  Hydrophobin fusions allowed aqueous 2-phase separations, for simple and rapid enrichment.  Inclusion of a Tobacco etch virus self-cleaving motif allowed removal of the hydrophobin.

hphobinThe “Perspectives” Session was notable for two talks, and a proposal: the latter was by Julian Ma for a “Society for Molecular Farming”, which was well supported and will probably kick off sometime this year.

Jim Larrick (Panorama Research, Mountain View, California) gave a typically eclectic, wide-ranging and highly enthusiastic talk on ‘Anti-fragility: Big picture issues in pharmaceutical development’.  He used the “Black Swan” analogy repeatedly to explain how the enterprise funding and pharma research sectors embodied fragile or anti-fragile thinking – with the observation that it was easier to resist black swans (eg: the unexpected) with a raft of small projects, than to have a few big ones.  He also pointed out that the NIH liked big projects – and that a useful alternative name for them was “Not Invented Here”!  Right up there with “Not Real Funding” as the alternative name for our National Research Foundation….

IMG_0133

Matthew Paul (St. George’s University of London) presented a set of 15 case studies of commercial paths to introducing molecular farming, which was very interesting to us academic types.  More interesting was the fact that while innovative and protectable technology and products were important to start-ups, the majority of successful ones had their basis in platform development – and the average time from platform to product identification was about five years.  Venture capital firms were considered too greedy for early-stage start-ups, but their involvement later led to stability as their partnering was long term.

Another interesting feature was that many of the successful ventures sold “side products”: for example, Ventria sold cytokines and cosmetic formulations, while KBP sold cell culture reagents.  Several also licenced out technology platforms, but the revenue was not held to be so good.

There were three main indicators of success:

  • Management quality
  • A good lead product
  • Having a panel of products

IMG_0135A good strategy to stay alive was “maximum income / minimum burn” – and he held up the example of Medicago in this regard.  He noted that in the absence of major investment from Big Pharma, Phase 2 trial success was the driver for commercialisation.

PBVAB 5 Verona June 2013: Session 7

3 August, 2013

Suzanne Huddy, a postdoc in our lab, kindly took some notes in a session I moderated at the 5th PBVAB in Verona this year.

Little did she know this is just my way of easing her in to doing this more often…B-)  Thanks, Sue!

Session 7: Manufacturing and Production Systems Developments

Moderator: EP Rybicki

Andreas Schaaf from Greenovation Biotech GmbH presented on “BryotechnologyTM en route to the clinic”, highlighting a production platform based on the moss Physcomitrella patens.  The overriding advantage of this system is that the moss is haploid and therefore genome modification is fairly straight forward with timelines for modifications similar to that of yeast systems.  Physcomitrella patens is also fairly unique since it has a very high occurring rate of homologous recombination (HR).  These traits along with the fact that the genome is sequenced and annotated allow fairly simple customization of the genomic background.  Using this, they have glyco-engineered strains and have removed plantized glycosylation completely.

Other than the products mentioned on their website (www.greenovation.com), they are currently working on α-galactosidase for treating Fabry disease.  Fabry disease is a rare genetic lysosomal storage disorder which results in the accumulation of lipids in the kidney, autonomic nervous system and cardiovascular system cells.  They are also working on the production of recombinant human β glucocerebrosidase for the treatment of Gaucher disease.  Interestingly, these are the same products produced by Protalix Therapeutics.

Stefan Schillberg from the Fraunhofer IME presented on “Co-MoFarm- Contained molecular farming: Controlled contained systems for high yield consistency”.  The CoMoFarm project has been funded for 3.5 years under the European Commission 7th Framework programme.  This project focused on the development of high-yielding plant-based production systems for recombinant proteins.

The presentation initially contrasted the production capability of the various plant platforms employed by this group using both HA (influenza hemagglutinin) and the human M12 antibody as protein products.  The production platforms included Arabidopsis and rice suspension cells, tobacco plants, roots and suspension cells, and moss suspension cultures.  The results presented highlighted the fact that one production platform is not necessarily optimal for all recombinantly expressed proteins, although the traditional tobacco leaves and BY-2 suspension cultures did produce the highest expression levels.  By further optimization of cultivation parameters (including media components), expression levels could be increased by up to 30 fold.  The presentation also showed that expression could also be improved by co-expression of the target protein with a fluorescent marker, DsRed.  In short, this allows the development of higher expressing lines through the non-invasive selection single elite expressing cells by flow-cytometry.  Stephan Schillberg also presented on the groups development of non-invasive monitoring systems for plant cell health and productivity.

The presentation was ended with a comparison on the cost of production of M12 antibody in either tobacco plants or BY-2 cells grown in 200 L bioreactors.  While the cost of producing this product in tobacco plants was less per gram of the product, the time for production in BY-2 cells was much shorter.  Details of the costing can be found at http://comofarm.org/useruploads/files/CoMoFarm_2013-6.pdf, where CoMoFarm have kindly made the presentation given in Verona available.

Pascal Drake from St. George’s University of London presented on “Hydroponic cultivation of tobacco for the production of recombinant pharmaceutical proteins by rhizosecretion”.  This presentation looked at the production and optimization of antibodies and Cyanovirin-N (CV-N) (a cyanobacterial protein which displays virucidal activity) in hydroponically cultivated tobacco plants.  Data was shown that suggested the inclusion of PGRs (plant growth regulators) and a nitrate source in the hydroponic medium could increase the concentration of the protein of interest in the medium.  Hydroponic cultivation has some advantages over traditional cultivation of tobacco plants.  Plants are cultivated in chemically defined media, therefore there is better control over the process and in this way this system approaches cell fermentation processes.  Additionally, fully processed secreted proteins can be harvested over the lifetime of the plant and purification can be simplified since the medium does not contain as many proteins as a whole leaf extract.  A “nifty” way of doing a western blot was also shown- basically, transgenic plants are germinated on nitrocellulose paper; this paper can then be used directly for a western blot since the protein of interest would have been secreted directly from the roots of the plant onto the membrane.  After development of the blot, the presence of the protein is seen in “root-shaped” pattern.

Bertrand Magy from the Institute of Life Sciences at the University catholique de Louvain, Belgium presented on the “Development of suspension cells as a competitive production system for antibodies”.  This research looked at designing an optimized antibody scaffold that can be combined with different variable regions in order to produce high levels of functional antibodies.  Initially, the expression of different IgG isotypes (human, rat and mouse) with the same variable region was investigated in tobacco and Arabidopsis thaliana suspension cells.  Bertrand showed that while antibodies accumulated in the extracellular medium, degradation occurred according to the isotype.  In this case, A. thaliana was also shown to be the better producer.  As is the case with many other cell suspension-based expression, the yield of antibody could be optimized by manipulating the growth medium.  Levels of antibody production of >30 mg/L could be achieved.

PBVAB 5 Part 2

28 June, 2013

Session: Vaccines 1.

This session produced some of the most interesting talks of the conference, so I will go into some detail in describing them.

Charlie Arntzen (ASU, Tempe, AZ) gave a typically excellent presentation on their latest work on norovirus vaccine formulation for stability and oral delivery – using lyophilized aloe gel-derived nanoparticles.

Norovirus outbreaks are tracked by a CDC lab continuously; every 2 years or so new strains circulate, meaning vaccines will have to keep up.  Ligocyte makes VLPs in insect cells currently; however, plant expression has been shown to be able to respond quickly to strain changes, via Icon vectors used at KBP, with the possibility of very quickly making a lot of product.  Downstream formulation has been a problem, however, as the processing throws away lots of antigen protein downstream.

Ligocyte use MPLA and chitosan (an irritant) for nasal immunisation: this has 50% efficacy.  The FDA does not like adjuvants for nasal dosing – so they went for no adjuvant, and chose the nasal route as one gets a more uniform response for 5x less Ag than with oral administration.  The formulation is basically of VLP preparations with lyophilized and milled pectin content from aloe gel: the uniformly-sized nanoparticles absorb water, and stick to each other and to the nasal mucosa for 3 hrs+.

Charlie commented that “This is the one time I recommend putting white powder in your nose!”.

They have tried mixing VLPs of different virus types – and found that with 50 ug of each, you get same immune response as to one.  Apparently this virus is unusual as you can do virus challenge experiments quite easily: these cost $15K/patient, which is a bargain.  The group is looking at annual or biannual dosing for maximum protection, and is also formulating VLPs for oral vaccination.  Interestingly, the aloe gel also works for intramuscular vaccination – possibly as a result of a depot effect?

 

Yuri Gleba (Nomad / Icon Genetics, Halle, Germany) was supposed to speak on “Technology progress in PMP (=plant-made pharma) research” – but basically said “Transient technologies are the future!”, and then went on to demonstrate it.  He noted that KBP can process 1.2 tonnes biomass/day for agroinfiltrating plants, using a robot from a car factory and a converted industrial autoclave – and consequently have to grow plants in trays for infiltration. Nomad had therefore started investigating how spraying Agrobacterium onto plants might work – with a biosafe Agrobacterium as a prime requirement.  They also took the bold approach of doing transient agronomic trait engineering – for traits such as flowering control, drought tolerance, yield suites, cellulases and anti-microbials – and sold the idea to Bayer Crop Science.

Their technique uses an engineered Agrobacterium that is 100-1000x more efficient at gene delivery than standard strains, with surfactants that allow easy penetration of the leaf tissues.  In combination with the use of replicating vectors that spread cell-to-cell, they could get 100% of standard infiltration yields, by a far easier and much more scalable technique.  They found that spraying worked for most dicots and even for maize, albeit inefficiently, and that they could repeatedly dose plants for the same trait with no apparent harm.  Transient expression of cry1ab and cry2ab Bt toxins delivery worked well, as did delivery of the Cold Shock protein from B subtilis, which also works for drought tolerance.

Their technique does away with need for seed – they can do somatic trait addition / subtraction, they can use the technology outdoors, and there is no trait transmission and so no escape, as the genes do not get into seed.  It means they can produce proteins in  plants at commodity agricultural prices – which considerably broadens the scope of “biofarming” in terms of what products can viably be made!!

One good example was cellulases for bioethanol production: one needs 1-3% w/w relative to cellulose mass, meaning production must be high volume and cheap.  Yuri noted it was possible to store biomass as silage or possibly by vacuum-packing at room temperature for months and that the silage process also eliminated Agrobacterium.  He mused that it might be possible to make a sauerkraut-type oral formulation for recombinant protein delivery…B-)

As for antimicrobials in plants, he said organic crops have more microbes than standard, eg: the recent fatal infections caused by E coli in bean sprouts in Europe – and that a solution would be to make eg colicin E1 in the plants, to kill the bacteria in situ.  One can apply for GRAS status which is MUCH faster than for other routes of approval.  They were currently doing this for phage lysins, bacteriocins, and thaumatin, among others.  Yuri said transient expression tech was like flash drives vs old-style PCs: a versatile set of tools vs a one-trick pony.  He also mused that the technology could lead to reinvention of the old ideal of use of biofarming in undeveloped communities – presumably for low-cost remedies as well as for therapeutics, etc.

To a question on what was the shelf-life of recombinant Agro he replied that there was already field use of live bacteria to combat pathogenic strains; that one can take a Petri dish and dilute in 100l water and spray, and then keep the suspension for two days…it was a very robust bug!  An interesting regulatory point that came up was that the USDA thinks a plant is a GMO even if it is transiently sprayed.

 

Andres Wigdorovitz (INTA, Buenos Aires, Argentina) spoke on their experience of a decade’s worth of work on plant-made veterinary vaccines.  He opened by noting that he has a major problem of getting money from companies in Argentina – partly die to what a “product” is defined as, because what happens is that a “researcher has an egg, whereas the company wants a butterfly”.

They made the decision to work in platforms – to make diagnostic kits initially, which teaches one how to make recombinant proteins.  They use baculovirus/insect cells and plants – in the form of transgenic alfalfa or transplastomic tobacco – to make the same proteins for comparison purposes, and as products, depending on which was more suitable.  While they had had considerable experience with FMDV vaccines made in plants, which had been protective, their current work focused on making novel vaccines and products.  An example was camellid-derived VHH nanobodies – and the fact that fusing the E2 protein of Bovine viral diarrhoea virus (BVDV) with a anti-E2 VHH gave a better alfalfa-produced immunogen for something that was already protective.  Their experimental vaccine was better than the commercial vaccine from the Ab response – and they could get total protection with 3 ug vaccine, and even better efficacy if they made an E2-HLA fusion.  He believed they will have a commercial vaccine in less than 2 yrs as they were engaged in getting regulatory approval now.

In other work, a FMDV VP1 peptide-GUS fusion expressed 10x better in transplastomic tobacco than in transgenic alfalfa.  A rotavirus VP8* fusion protein was also 10x increased in chloroplasts, and dry leaves preserved the protein very well.  They were also making VHH nanobodies against human rotavirus as vaccine coverage of local strains was not good – and VHH against the conserved VP6 could penetrate the outer capsid and bind and neutralize infectivity whereas larger proteins did not work.  They got 3% TSP expression in tobacco chloroplasts.  They were also making VHH to other rotavirus proteins, and to human noro- and influenza viruses.  All in all, it was a very heartening demonstration of a good business model, and that developing countries too can lead the field in some respects.

 

The remainder of the session was taken up with two talks from our group: these were given by Drs Ann Meyers and Inga Hitzeroth, on the parrot-infecting Beak and feather disease virus CP-elastin fusion protein production, and Human rotavirus CP and VLP production in N benthamiana via agroinfiltration, respectively.

The BFDV work has just been published with MSc student Lucian Duvenage as first author – from PubMed, then:

J Virol Methods. 2013 Jul;191(1):55-62. doi: 10.1016/j.jviromet.2013.03.028. Epub 2013 Apr 9.
Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides.
Duvenage L, Hitzeroth II, Meyers AE, Rybicki EP.
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7700, South Africa.

Abstract

Psittacine beak and feather disease, caused by beak and feather disease virus (BFDV), is a threat to endangered psittacine species. There is currently no vaccine against BFDV, which necessitates the development of safe and affordable vaccine candidates. A subunit vaccine based on BFDV capsid protein (CP), the major antigenic determinant, expressed in the inexpensive and highly scalable plant expression system could satisfy these requirements. Full-length CP and a truncated CP (ΔN40 CP) were transiently expressed in tobacco (Nicotiana benthamiana) as fusions to elastin-like polypeptide (ELP). These two proteins were fused to ELPs of different lengths in order to increase expression levels and to provide a simple means of purification. The ELP fusion proteins were purified by inverse transition cycling (ITC) and it was found that a membrane filtration-based ITC method improved the recovery of ΔN40 CP-ELP51 fusion protein relative to a centrifugation-based method.

Essentially, Lucian managed in some very elegant work to show that BFDV CP fused to a 51-mer ELP allowed production and subsequent simple purification of quite high yields of fusion protein.  It remains to be seen how immunogenic or protective this is – however, it is a breakthrough, as expression of the CP alone has been VERY problematic, in everything from insect cells through E coli, to plants.

Inga spoke on our recent MSc student David Mutepfa’s work on expression in plants of the CPs of a South African rotavirus that is not well matched to current live attenuated vaccines.  The short story is that he succeeded very well indeed in expressing three of the four proteins.  From a recent publication from me and Nunzia Scotti on plant-made VLPs, then:

Current studies in the Rybicki laboratory have focused on expression of capsid proteins of a local isolate of human rotavirus (G9 P[6]) that is not well matched to available commercial vaccines.  Expression of VP2, VP4 and VP6 in N. benthamiana was targeted via co-agroinfiltration to the cytosol, endoplasmic reticulum, apoplast and chloroplast. Electron microscopy showed that co-expressed VP2/6 and VP2/6/4 produced virus-like particles in the cytosol, with yields as high as 1.1 g/kg of plant material, for batches of 100 g.

…with a picture to prove VLPs are made:

rota pic

 

Rotavirus VP2/6/4 co-expression in N benthamiana: protein ex- tract partially purified by sucrose gradient centrifugation, particles captured onto electron microscope grids with mouse-anti VP6 antibody. Bar = 200 nm


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