Posts Tagged ‘biopharming’

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-)

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.

Trade Secrets: Are Green Vaccines Appropriate for Africa? : Trade Secrets

21 June, 2012

See on Scoop.itVirology News

I have mentioned several times here, and elsewhere, that my lab works on expressing vaccine-relevant viral proteins in plants – and that I think this is a highly appropriate technology for the purpose.  Read more…

See on blogs.nature.com

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

Background
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.

Results
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.

Conclusions
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.

 


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