Posts Tagged ‘plant-based’

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.


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.


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…!



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


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).


Watch this space…B-)

Plant-Based Vaccines, Antibodies and Biologics 5: Part 1

27 June, 2013

Plant-Based Vaccines, Antibodies and Biologics: the 5th Conference

Verona, Italy, June 2013

The return of this biennial meeting to Verona – the third time it has been held here – was a welcome change; while the previous meeting in Porto in 2011 may have been good, the city was nothing like as pleasant a place to relax.  My group is now familiar enough with Verona that we know just where to go to get pasta by the riverside – or, on this occasion, “colt loin with braised onion and potatoes” and “stewed horse with red wine”.  Which seem more palatable, somehow, as “Costata di puledro con cipolle brasate e patate” and “Stracotto di cavallo speziata” respectively, but were enjoyed anyway.

The conference kicked off with an opening plenary session, chaired by the Local Organizing Chair, Mario Pezzotti, of the University of Verona.  The headline act was a talk on taliglucerase alfa – aka glucocerebrocidase, a Gaucher Disease therapeutic  –  by Einat Almon of Protalix Therapeutics from Carmiel, Israel.  I featured the product here last year, after an earlier feature here; suffice it to say that it has soared since FDA approval, and now Protalix is pushing hard with new plant-made products to follow it up.  While they use carrot cells for taliglucerase alfa, apparently they are using suspension-cultured tobacco cells for other products – and are using an easily-scalable disposable 800 litre plastic bag system, with air-driven mixing of cells suspended in very simple, completely mammal-derived product free media.  Hundreds of patients had been treated with the drug for up to 5 years with no ill effects, and the possibility of switching therapies from mammalian cell-made products to the plant cell-made had been successfully demonstrated.

Scott Deeter of Ventria Biosciences (Ft Collins, USA) spoke next, on “Commercializing plant-based therapeutics and bioreagents”.  His company has possibly the most pragmatic attitude to the production and sale of these substances that I have yet met, and he struck a number of chords with our thinking on the subject – which of course, post-dates theirs!  Ventria use self-pollinating transgenic cereals for production of seed containing the protein of interest, and rice in particular, for safety reasons – and because the processing of the seeds is very well understood, and the purification processes and schedules are common to many food products and so do not require new technology.  He reckoned that a company starting out in the business needed an approved product in order to give customers confidence – but should also engage in contract services and contract manufacture of client-driven products in order to avoid being a one-product shop.  To this end, they had received APHIS Biological Quality Manufacturing Systems (BQMS) certification (similar to ISO9001), with the help of the US Biotechnology Regulatory Services.

Their therapeutic products included diarrhoea, ulcerative colitis and osteoporosis therapeutics which were already in phase II clinical trial.  Scott noted that in particular, recombinant lactoferrin was a novel product, which could only feasibly be produced in the volumes and at the price required for effective therapy, by recombinant plant-based production systems.  It also filled a high unmet need as a therapy for antibiotic-associated diarrhoea in the US, with +/-3 million patients at risk annually who presently cost service providers over $1500 each for treatment.

A third commercialization option was bioreagents and industrial enzymes, which they marketed via a vehicle called InVitria: they had a number of products already in the market, which Scott claimed gave confidence to the market and to partners, while building capacity to make therapeutics.  Something that was particularly attractive to our prospects was that a collection or pool of small volume products – say $5-10 million each – gave a respectable portfolio.  He noted that Sigma Aldrich and Merck were already marketing their human serum albumin, which competed effectively with serum- and yeast-derived products.

George Lomonossoff from the John Innes Centre in Norwich, UK, spoke next on “Transient expression for the rapid production of virus-like particles in plants” – a subject close to our hearts, seeing as we have for the last five years been associated with George and partners in the Framework 7-funded PlaProVa consortium.  He mentioned as an object example the recent success in both production and an efficacy trial of complete Bluetongue virus (BTV) serotype 8 VLPs, made in Nicotiana benthamiana via transient expression using their proprietary Cowpea mosaic virus (CPMV) RNA2-derived pEAQ vector: this was published recently in Plant Biotechnology Journal.

Another very useful technology was the use of CPMV capsids as engineered nanoparticles: one can make empty VLPs of CPMV at high yield by co-expressing the coat protein (CP) precursor VP60 and the viral 24K protease: the particles are structurally very similar to virions in having a 0.85 nm pore at 5-fold rotational axes of symmetry, meaning they can be loaded with (for example) Co ions.  It is also possible to fuse targeting sequences – such as the familiar RGD loop – into the surface loops of the CPMV CPs, and to modify the inner surface too.  One application would be to engineer Cys residues exposed on the inside, which could bind Fe2+ ions: this would result in particles which could be targeted to cancer cells by specific sequences, then heated using magnetic fields.

John Butler of Bayer Innovation GmbH (Leverkusen, Germany) closed out the session with an account of lessons learned from the development of the plant-derived non-Hodgkins lymphoma (NHL) vaccine, that they had acquired with Icon Genetics, who in turn had inherited it from the sadly defunct Large Scale Biology Corp.  It was rather depressing to hear that Bayer had dumped the vaccine, despite the developers having reached their targets in turning 43 of 45 tumour samples into lifetime individualized supplies of vaccine within12 weeks, and despite the phase I trial being as successful as could be hoped.  To this end, the vaccines had been well tolerated and were immunogenic; of the patients who reacted immunologically, all but one were still tumour-free presently.

He felt that the problem was that NHL trials were too long and therefore too expensive as it was a slow-progressing disease; that a different clinical approach was needed, and that using the vaccines as a first-line therapy instead of only after the 2nd or 3rd relapse would be a much better idea.  The main lesson learned was that proving the technology would be far better done with a therapeutic vaccine for a fast-acting cancer, which would allow 1-2 year clinical trials with overall survival as an endpoint.

(more coming)

Setting up a platform for plant-based influenza virus vaccine production in South Africa

5 May, 2012

A virus-like particle formed by influenza virus haemagglutinin budding out of plant cells. By Russell Kightley Media

See it also on Scoop.itVirology News

Our (very) recently-published article on plant-made flu vaccines in BMC Biotechnology:

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Elizabeth Mortimer, James M Maclean, Sandiswa Mbewana, Amelia Buys, Anna-Lise Williamson, Inga I Hitzeroth and Edward P Rybicki

During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy.

For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses.

We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.”

I have mentioned time and again that going green is the sensible thing to do: here is a concrete example of how my research group is trying to go about it.  This is a very sensible technology for rapid-response vaccine production, and especially for emerging or orphan or pandemic virus threats.  We got really good expresion levels of H5N1 HA protein via transient expression in plants, and have already started on pandemic H1N1 HA expression.  Let’s hope some governmental types in SA take some notice!

I thank Russell Kightley Media for the specially-commissioned graphic of budded HA-only VLPs.