Archive for the ‘Vaccines: General’ Category

“Controversial scientist recreates H1N1 flu that killed 500K people” – NOT

2 July, 2014

Dr Yoshihiro Kawaoka, professor of virology at University of Wisconsin at Madison, has tweaked the 2009 strain of pandemic influenza to make it resistant the human immune system’s antibodies.


Trust the Dimwitted Mail to misstate what happened – which is that Yoshihiro Kawaoka selected the H1N1pdm 2009 flu virus in culture till he came up with antibody-binding escape mutants.

What he said:

‘Through selection of immune escape viruses in the laboratory under appropriate containment conditions, we were able to identify the key regions [that] would enable 2009 H1N1 viruses to escape immunity,’

Now recall that the H1N1pdm 2009 virus is NOT a particularly nasty variant; that it has NOT been proved the escape mutants will infect vaccinated people at all – and that all the work was done "a state-of-the-art laboratory at the Institute for Influenza Virus Research in Madison", so the odds that it will get out are VERY low.

But papers have been sold, and the scare is in.

HPV type 16 E7 protein bodies cause tumour regression in mice

26 May, 2014

See on Scoop.itIIDMM News


Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer.


In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV – type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera(R), a self-assembly domain of the maize gamma-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems.


High-level expression of the HPV 16E7SH protein fused to Zera(R) in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression.


The fusion of 16E7SH to the Zera(R) peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera(R) PBs.


I thank Russell Kightley Media for use of the HPV/cervical cancer graphic

Ed Rybicki‘s insight:

I keep saying – you gotta go green…B-) And here we are, suiting action to words.  

Modestly, of course.  

Well done to Mark Whitehead and Thomas Oelschlager; thanks to Inga for sticking with a difficult ms – and thanks Era Biotech for the technology!

See on

Recombinant Bluetongue virus vaccines – or some, anyway

1 May, 2014

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

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

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

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

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

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

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

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

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

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

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

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

Antivaxer HPV nonsense!

14 March, 2014

Fair Lady magazine just published a short but very useful article on HPV vaccines, with question-and-answer format commentary from a number of local experts on safety and such.

Then some loony takes them on in this piece.

My comment to that:

OK: this is a sadly under-informed and over-complicated response to a very good, simple article, with expert comments that are no way out of line with what is known to be the case about these vaccines.

Please allow me to correct a few things: in the first place, you say “Several ingredients in the two HPV vaccines are known to be a problem. One is the use of the microbe Saccharomyces cerevisiae, common yeast, as the medium in which the Gardasil antigen is developed. S. cerevisiae is known to trigger autoimmune response, as discussed recently in Yeast in Vaccines Tied to Autoimmune Diseases. Cervarix, though, was produced with a different medium, Trichoplusiani.”

Yeast is known to trigger autoimmune responses? Really? Then what about all those millions of people who have received Hepatitis B virus vaccine, also made in yeast? There is NO documented correlation with vaccination with yeast-made products and autoimmunity. NONE.

Then, Cervarix is “made in a different medium” – you obviously mean cell type, because Trichoplusia ni [note!] is a species of insect, from which the tissue-cultured cells used to make the HPV vaccine are derived. And? You have no problem with that? You shouldn’t.

Further: “The two vaccines, Gardasil and Cervarix, are distinctly different in another way. Gardasil contains a single adjuvant… while Cervarix utilizes a combination …These differences, since they involve the hyper-activation of the immune system and a known trigger for autoimmune disorders in only one of the vaccines, suggest that a recent study’s finding that there are no adverse effects whatsoever in either vaccine beggar belief.”

So you have a problem with the finding that there are no adverse effects BECAUSE the adjuvant regime is different? Not because of the evidence? Sorry, that isn’t very good science!

And it gets better: “Most significantly, in every clinical trial evaluating safety for both Gardasil and Cervarix, the so-called placebo groups were given injections that included an active aluminum adjuvant!
Though this is a common practice in vaccine trials, it is obviously a blatant means of biasing the results.”

So – a perfectly acceptable placebo arm in a clinical trial, with adjuvant given so that any difference would not be due to just this, means the studies are meaningless? Really?? What about the UNvaccinated groups these were compared to? ALL vaccines are associated with SOME events – and I note that YOUR piece above contains the sentence “The high proportion of adverse events reported is mainly due to the design of the study, since women were requested to report all events occurring after the vaccination; however the majority of events were mild and transient”.

You then quote – at excruciating length – a number of articles purportedly supporting your case, that can be summarised as showing that “HPV vaccination does not have a therapeutic effect in young women with pre-existing human papillomavirus infection”.

Yes? And? The two licenced HPV vaccines are PROPHYLACTIC, and were never intended to be therapeutic! It is unfortunate that the vaccines do not in fact lessen ESTABLISHED infections; however, there is plentiful evidence that they PREVENT INFECTIONS FROM BEING ESTABLISHED.

You are being alarmist and spreading falsehoods from a very shaky evidential base. You should stop doing that, or risk being shown up as being an antivax crank.

Eat your vaccines

7 February, 2014

See on Scoop.itVirology and Bioinformatics from

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

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.


Legends alive: from left, Fred Murphy; Daan Verwoerd; Bob Millar; Henk Huismans; Errling Norrby; Marc van Regenmortel

Jean-Marie Andrieu; Marc van Regenmortel – at a VERY good unofficial dinner


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.


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-)

Bats and SARS-CoV: deja vu all over again

1 November, 2013

In 2008, I wrote a blog piece entitled “Who do you bind to, my lovely?”, about a couple of papers on SARS-CoV – the coronavirus that causes severe acute respiratory syndrome.  I closed that piece with the following:

“Adding fuel to the speculative fire is another paper in the same issue: this reports that there is evidence of a recombinant origin for SL-CoVs, and there is probably “…an uncharacterized SLCoV lineage that is phylogenetically closer to S[ARS]CoVs than any of the currently sampled bat SLCoVs.”

So let’s all just wait for the next one, shall we?”

…in connection with the fact that horseshoe bat coronaviruses were VERY similar to SARS-CoV, but bound to different receptors.  And we had to wait five years, but heeeeeere we are… these folk may well have found the missing virus(es) that are directly transmissible from bats to humans:

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

 Xing-Yi Ge et al.

Nature (2013) doi:10.1038/nature12711

Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far [! - my comment; the last ones were pretty close...] more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

So – I told you so…B-)


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.


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-)


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