Posts Tagged ‘antibodies’

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-)

PLoS Pathogens: ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge

24 August, 2012

See on Scoop.itVirology and Bioinformatics from

“Here we show that live-attenuated SIV induces progressive increases in ADCC over time, and that the development of these antibodies is dependent upon the persistent replication of the vaccine strain. In two different experiments, the animals immunized with live-attenuated SIV that remained uninfected after pathogenic SIV challenge had higher measures of ADCC than those that became infected. Our results suggest that antibodies contribute to protection by live-attenuated SIV, and that persistent stimulation of antibody responses may be essential for HIV-1 vaccines to induce high ADCC activity.”


Shit HOT results, in that they demonstrate that – as some have said repeated ly over years – that neutralising Ab are NOT necessarily the Holy Grail, and that ADCC and other mechanisms are also really important.  Good Stuff…B-)

See on

HIV Vaccines From Bangkok – 3

20 September, 2011

HIV: a retrovirus. Courtesy of

The Wednesday morning agenda for the conference followed a somewhat bemusing Tuesday evening entertainment: one day I will learn NOT to involve myself in anything that involves getting onto a fleet of buses in the company of several hundred other people, and especially not in Bangkok!  It took us one-and-a-half HOURS to go from the venue to the Navy Yard for a reception and supper – but the first half an hour was spent just going around the block, such is the traffic density at rush hour.  There followed the standard fare for a conference in any country with any sort of culture: local entertainment (drummers and folk running around with bolts of cloth in this case), together with so-so food with very little choice, too much noise, and no possibility of being heard more than one person away.  But thankfully, only a twenty minute ride back!

“New Prevention Strategies” was the theme for the second set of plenaries – which were opened (unexpectedly; she was second on the programme, but No 1 overslept) by our very own Carolyn WIlliamson (IIDMM, UCT), speaking on implications for combination prevention strategies from HIV pre- and post-infection studies.  Carolyn noted again a point first raised by Pontiano Kaleebu on the first evening, that future vaccine efficacy trials should as as a matter of ethics offer preventions – eg ARVs – as a minimum standard of care, which will affect size and expense as well as endpoints like acquisition and disease progress.

She pointed out that 80% of infections with HIV were due to single viruses – but 20% were due to multiple infections, influenced by dose, IV injection, MSM transmission, inflammatory genital tract infections and the like.  The lesson from study of the Phambili and STEP trial breakthrough infections by sieve analysis showed vaccination had had a selective effect on T-cell pressure.  Phambili got 277 sequences from 43 people, in vaccine and placebo arms.  The Merck vaccine had no effect on the transmission bottleneck.  Scanning sites across the genome showed 2 sites of selection in Gag and 1 site in Nef were significantly different in the two arms; one in the region p6 of Gag looks like an epitope escape.  There was a weaker signal in Phambili than in STEP, however: this was due in part to the lower number of participants (the trial was stopped before recruitment was complete), the fact that there were men and women involved vs mainly men in STEP, among other factors.

It is worth remembering that the Phambili and STEP trials were stopped in 2007, and reported on at a very gloomy AIDS Vaccine Conference in Cape Town (covered here in ViroBlogy).

There was also no effect of pre-exposure Tenofovir on the transmission bottleneck, or evidence of immunity in highly exposed uninfected individuals using the gel – despite the evidence for “chemovaccination” in macaques, due to abortive infection checked by ARVs in target cells.  Thus, chemovaccination did not enhance the of impact microbicides and preinfection immune responses would not interfere with vaccine monitoring by this assay.

Tenofovir did impact early Ifn-g Gag-specific CD4+ responses post infection – indicating that possibly the drug prevents the initial destruction of CD4 cells in the gut, which would be a very valuable result.

Carolyn finished by noting that the implication for combination of preventive therapies is that it will increase the complexity of trials, make them cost considerably more, and make them longer.  However, microbicides that reduce inflammation may dramatically reduce infections, ARVs may increase the barrier to infections, and also increase the time for the effects of vaccination to kick in and increase post infection immunity, and combination of multiple partially effective interventions may have significantly greater impact than any alone.

Helen Weiss (London School of Tropical Medicine and Hygeine) was the late riser: she spoke on lessons from male circumcision for other prevention strategies.  It was interesting to many of us that it was a study in Nairobi in 1989 that showed the effect first – circumcision protected to some extent against in infection even in the presence of genitourinary diseases (GUDs).  A metastudy combining 15 studies subsequently showed reduced risk in all, to a 60% protection level.  Accordingly, three studies had been set up in Uganda, Kenya and SA  in 2005-2007 to directly study the effect.  They saw 50% efficacy in all locations, and all were stopped early as there was an obvious effect, with all participants being offered circumcision.  The studies saw an overall 58% protective effect, and the  effect in the Uganda trial persists up to 5 years post trial.

As for why this should be, Helen said that some studies say that the inner foreskin has a greater density of Langerhans and T-cells compared to the outer – and there is some evidence the inner is more easily infected in explant studies.  HIV infections also induce retention of Langerhans cells within the epidermis of the inner foreskin.  There is evidence that the inner foreskin facilitates efficient entry and translocation of cell-associated HIV, retention of Langerhans cells, and the incidence of infection is greater in men with larger foreskin area.

The conclusion was that one should offer circumcision in HIV prevention studies where heterosexual contact is the mode of transmission.  Among MSM, habitual penile inserters show some effect of protection, while habitual accepters are obviously not protected.

She closed by commenting that scaling up circumcision to 80 % coverage of adults and newborns by 2014 could save US$ 40 billion US: however, the reality was that uptake was slower than planned, with only 2.6% done by 2010.  However, there was obvious buy-in with a fourfold increase in circumcisions between 2009-2010.

While I thought she oversold the intervention rather – it is decidedly less simple than drug or microbicide interventions after all, benefits only one partner directly, and the lesson in South Africa is that even communities with a high circumcision rate can have very high prevalences of HIV infection – there is no doubt that circumcision in combination with pre- and post-exposure ARVs and microbicides cannot do other than have an additive effect in protection, and possibly even a synergistic one in some cases.

For me that was the morning; I missed three very worthy parallel late morning oral sessions while dealing with nagging emails – but started fresh again in the post-lunch period (an aside: best conference coffee break munchies and light lunches I have ever seen…B-), with Oral Session 11 – Mucosal Immunity.

Anthony Smith introduced us to a fascinating study of transcriptional “imprints” correlating with protective immunity in macaques following vaccination with the live attenuated SIV-∆-Nef virus, done by microarrays on RNAs from the cervix.  Smith noted that live attenuated viruses offer some of the best protection available in monkeys, and that SIV Mac239-∆-Nef was one of the best.  They isolated total RNA ex the cervix of rhesus macaques post-challenge with a heterologous virus at 140 days with native virus and tested unvaccinated and vaccinated samples with an Affymetrix rhesus chip.  There was 103-fold less viral RNA in vaccinated animals, and very little overlap of gene expression – only 1% (eg 5 genes) – of 405 vs 246 unvaccinated to vaccinated samples.  There was greater expression of inhibitors of innate immunity and inflammation in vaccinees; MIP3alpha expression was higher in unvaccinated monkeys – this brings in effector cells, including CD4+ T-cells, which would enhance infection.  Unvaccinated monkeys get a signalling cascade of cytokines which cause an inflammatory response – vaccinees get a short circuit in this signalling by mucosal conditioning with mutant virus.  There were important differences in humoral responses too, which were not reported here.  In light of this one could almost wish that the proposed trial in humans in the 1990s of the natural Nef deletant HIV-1 found in Sydney and associated with long-term non-progression had gone ahead – but only almost, as people with the virus did eventually start to progress to AIDS.

Steve Reeves then spoke on mucosal natural killer (NK) cells in SIV infected monkeys in chronic infection: he noted that NK cells respond early in infection in a variety of tissues.  They act to suppress viral replication in vitro, and are linked to disease control in vivo.  while much of what he said was straight over my head – I really do not have much truck with cytokine signalling cascades and lymphoid cell types and subtypes – it is becoming increasingly evident that not only are NK cells actively involved in controlling HIV infections, but that there are hitherto unsuspected variations among them, and often evidence of specificity in their interaction with infected cells.  Expect to hear much more about these fascinating guys in the future….

There followed a slightly disappointing talk by Shari Gordon, on the use of Human papillomavirus (HPV) pseudovirions (PsV), made in cell culture from co-expression of transfected HPV L1 and L2 capsid proteins and a replicating plasmid vaccine, to immunise mice vaginally.  The idea was to use a mucosa-infecting agent to make a vaccine which induces T-cells and antibody responses at mucosal sites to prevent HIV infection, during the window of opportunity where founder infections are being established.  HPV naturally infects the disrupted vaginal mucosa via interactions of L1 and L2 with receptors on basal keratinocytes – thus it was necessary to disrupt the vaginal epithelium by administration of progesterone and the known inflammatory agent nonoxynol-9 in order to infect.  They used HPV-16 PsV vectoring a SIV gag gene, then boosted with non-cross-reacting HPV-45 PsVs, both expressing red fluorescent protein (RFP) as a marker for in vivo fluorescence tracking.

They got a good response to HPV, and see anti-Gag IgA in vaginal secretions and IgG in serum.  They also see recruitment of T-cells to the site of infection in mucosa – both CD4 and 8 and activated cells.  T-cell responses assayed by intracellular cytokine staining  (ICS) showed that they get CD4+ and 8+ cells in tissue and in blood, which waned over time.  They then set up an experiment to see if systemic priming and mucosal HPV PsV boosting could protect macaques, using a regime consisting of sequential HPV-16, -45 and -58 PsV administration, with and without ALVAC + gag, and gp120 administered with the PsV 45 and 58.  They saw the same gp120 titres at the end of the regimen, with or without ALVAC priming.  They got a Gag-specific response, which expands and recruits T-cells in the genital tract but was lower in blood.  The response was better with ALVAC priming.  There was a primarily monospecific response of both CD4 and 8 T-cells, and primary effector memory.  Upon SIV challenge they saw a similar rate of acquisition despite the immune responses – however, they were only in mid-experiment, and still hoped to see viral control.  She noted the vaccine does not exacerbate the SIV infection rate.

While this was all good science, it was disappointing for a number of reasons.  First, they did not do or report the obvious control, of using DNA only in parallel with PsVs.  Second – in the opinion of my resident HIV/HPV vaccine expert, Anna-Lise Williamson – such vaginal immunisation using PsVs in humans would be a complete non-starter, because it is not ethically acceptable to use agents like nonoxynol-9, which is known to increase HIV infection rates, in a vaccine regimen.  Third, the vaccine did not seem to be very good, despite the supposed advantage of using particles to deliver a DNA vaccine: this is a subject close to my heart, given an interest in both HPV VLPs and DNA vaccines, and I think that oral or intranasal immunisation would have been a far better idea.  Fourth, and although this was not stated, the PsVs are made in immortalised 393TT cells expressing significant amounts of an oncogenic viral protein (polyomavirus T antigen) to enable replication of the vector plasmid – all of which I am sure would be a stern no-no for use in humans.

H Li spoke on the use of recombinant adenovirus vectors in monkeys: he noted that effector memory cells were induced by replicating viruses while non-replicating induced primarily memory cells in blood.  However, people had not looked at mucosal responses.  Accordingly, they used single or double recombinant Ad26 immunisations and showed one could get mucosal T-cells.  With a heterologous Ad5/26 prime/boost they get a potent and widely distributed T-cell response, which they have followed for 4 yrs and still see the responses 2.5 yrs post boost.  Mucosal T-lymphocytes are persistently activated.  They looked at T-cells in PBMC vs colon, duodenum and vaginal tissue: the latter were activated while PBMC were not, so there was only transient activation here.  Memory phenotype shows Tem (effector memory) to Tcm (core memory) evolution in the periphery.  Mucosal T-cells show a persistent Tem1 phenotype.

Ming Zeng revisited the attenuated live SIV vaccine, and its mucosal protective properties.  Live attenuated vaccines offer the best protection yet in monkeys against homologous or heterologous virus challenge – and understanding the correlates would help understand design principles for human vaccines.

They inoculated monkeys with SIV-∆-Nef intravenously, and challenged with repeated intravaginal inoculation.  He showed evidence of a fascinating vaccine-induced Ab concentration at the mucosal border of the monkey cervix, correlated with limited spread and prevention of infection.  They cannot see significant challenge viral growth at portal of entry in vaccinees from 20 weeks post vaccination.  Tissue-associated IgG is concentrated at the port of entry at 20 wk in the cervix and vagina: distribution of the IgG shows one gets plasma cells at the cervix, but also IgG-staining cells especially just underneath the epithelial cell layers.  The cells are epithelial reserve cells and enrich IgG inside cells, presumably by uptake mediated by the neonatal IgG receptor expressed on their surfaces.  This can be shown in vitro by incubating plasma cells with a filter-separated layer of epithelial cells from the female reproductive tract (FRT).

In challenge phase they noticed Ab concentration increased rapidly after challenge in situ.  All genes involved in Ab synthesis were upregulated in challenged monkeys in FRT and germinal centre cells see a dramatic local expansion of plasmablasts after challenge – presumably of memory B cells.

They think the SIV-∆-Nef vaccine converts the FRT to an inductive site for B cell expansion and maturation.  They get 5-10x the amount of IgG produced vs IgA.  They think it is both local recruitment of B cells and activation of local cells that results in the IgG production – which is total and not just HIV-specific IgG.

Again, this is a fascinating result obtained using a controversial vaccine candidate – and one which is not going to go away.

Late afternoon Wednesday was the turn of Symposium Session 02: Recent Advances in B Cell & Protective Antibody Responses – and two talks that took the prize as far as I was concerned were one by Peter Kwong and the following one by Pascal Poignard, both from Scripps in San Diego.

I couldn’t pretend to do justice to the Kwong talk: the graphics were so good, and there was so much detail, that it was like watching a great big complicated shiny machine in motion.  It was very beautiful, but I couldn’t tell you exactly what it is that he did.  Suffice it to say that he introduced us to the concept of mining the “antibodyome” by using structural bioinformatics to get solutions for vaccines by deep sequencing.  A consequence of this was that they could follow the maturation path of specific clones of cells making antibodies binding specific Env epitopes.  An important thing to come out of his talk was a possible reason for why strongly-binding broadly-neutralising antibodies are so rare: they found that, for their preferred target of the CD4 binding site, initially-produced antibodies were of very low affinity and needed a lot of maturation to become strongly neutralising and broadly reactive – which, of course, meant the producing cells were generally selected against and did not make it to being memory B-cells.  Knowing what was possible, however, and being able to make antigens to stimulate those antibodies specifically, would make for a rational vaccine design strategy.

Pascal Poignard described something that has been much in the news lately: the recent discovery of many strongly-binding broadly-neutralising monoclonal antibodies in people living with HIV.  He detailed how the IAVI protocol G search screened 1800 donors, mainly from Africa, for “elite neutralisers”.  They took the top four and did high throughput screening of memory B cells with antigen, and rescued the Ab gene sequences from selected wells, triaged them, and ended up with a selection of potent neutralising MAbs.  These were mostly broadly neutralising, but some were very potent – tenfold better than the previous best.  One group of 5 MAbs – all from the same individual – bind at various sites around the V1 and 2 and 3 loops of Env; another group of 3 from different individuals bind glycans and the V3 loop.  Data suggest protection needs 100x the IC50 value – which was very low for most of them, meaning they could be highly efficacious at low concentration, and synergise each other’s effective in mixtures.  Certain combinations of MAbs would give better protective coverage than others – especially if they did not neutralise the same spectrum of viruses.

Sprawling Bangkok - from the 37th floor

The work raises all sorts of very interesting possibilities, including mimicking the structures bound so well by these MAbs in order to elicit them more frequently, as well as using them therapeutically or in prevention regimes.  As far as antibodies are concerned, it is apparent that we are in a new era of sophistication as regards the potential for both exploiting the natural “antibodyome”, and even designing our own.

There followed a most enjoyable “Faculty Dinner” – my wife got me invited – on the 54th floor of the Centara Grand Hotel, followed by an even more enjoyable sojourn with pleasing beverages on the open deck of the 55th floor, overlooking Bangkok.

Until it rained, anyway.