I am very grateful to the previous speaker, Luc Hessel, for setting me up so nicely, because I think right at the end of his talk he basically said that we can’t do what I am trying to do!
The other thing I am indebted for, more seriously, is that Luc Hessel highlighted the manufacturing challenge that we face in terms of vaccines, and in particular the difficulty of changing a process scale. Chemical engineers can build a chemical process by sitting behind a PC. Biological systems are much more complex, and in manufacturing it is that biological complexity, interacting with physical effects as you change your process scale, that control the outcome and hence the nature of your product.
There is an added complexity – which I am sure most of the excellent immunologists in the audience know far better than I do – that the immune system then amplifies that variability in your product. Consequently you get a cascade of complex biological-physical interactions in a process, leading to a bad immunological outcome if you are not careful and if you don’t have good control over what you are doing.
The rationale at the Australian Institute for Bioengineering and Nanotechnology (AIBN), as one side of what we do there – and I have several excellent colleagues working with me on these sorts of things – is to try to understand how better to do industrial bioprocessing, and indeed process scale-up.
The particular area that I am interested in, or one of them – and I almost feel I should be talking about my large-scale peptide manufacturing work today, having heard a number of talks on peptides – is the large-scale processing of virus-like vaccines, or virus-like particle vaccines.
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I certainly don’t need to tell this audience that vaccines have had a huge impact on health. However, I am putting these pictures up not because of the vaccines but because of the way in which the vaccines have been manufactured. The two on the left there – representing Jenner’s and Sabin’s processes – do not refer to particularly good manufacturing technologies, although those processes did make rather efficacious vaccines. And of course the Salk/Enders process is the cornerstone of the modern pharmaceutical industry at the present.
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To look specifically at virus-like particles (VLPs): these are an emerging vaccine class showing some interesting effects. We have, of course, seen hepatitis B already.
More recently the HPV 16 vaccine for cervical cancer, developed by Professor Ian Frazer, has shown some unique properties, I think, in terms of vaccines. It is more or less 100 per cent immunoprotective, for selected major strains. If one looks at how it is produced, one sees that essentially it is made as protein in yeast, these protein particles are disassembled, purified and then reassembled for formulation and injection. And, as I say, it is highly immunoprotective. That seems to result from the particulate structure, and it seems to be an emergent theme in this field that the adjuvants are the particles themselves, that presentation of structure is critical for good immune reaction.
In terms of emerging seasonal and pandemic influenza vaccines, there is a lot of discussion about these inactivated whole viruses being self-adjuvanting. There have been some academic studies by Merck in taking the HPV 16 particle and, essentially, chemically conjugating peptides onto that from the matrix protein of influenza, which gave very good results in a mouse challenge model. (Of course, I’m aware mice lie!)
More recently, a three-protein virus-like particle has been developed by Novavax. It is highly immunoprotective, and is delivered nasally. Also, a ‘universal’ VLP vaccine, again based on that M protein peptide from influenza, has been developed as a HepB chimera. And there are some gastroenteritis VLPs under development.
So there is an emerging class of products that I, as a biochemical engineer or a bioprocess engineer, am not sure how to manufacture robustly. That is what, essentially, drew me into this field.
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So what is a virus-like particle? Well, here is a virus.
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The particle is essentially a protein shell with a viral genome in it. However, the virus particle lacks the viral genome and hence is not infectious. It is essentially the protein shell without the infectious bit.
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So how are VLPs made? There are different process routes. The one I am going to zoom in on is really the process that Merck uses to make this cervical cancer vaccine.
They start essentially by expressing precursor proteins within yeast, purifying these, and doing an in vitro assembly to the product. The advantages of this are, firstly, that you have a very efficient protein manufacturing approach. Protein therapeutic manufacture has been around now for 20 to 30 years, and industry can do it rather well – much more easily, in some cases, than making whole-virus vaccines. After making the protein, they assemble it as a VLP. So essentially it gears the industrial knowledge that has already been in existence, in terms of safe protein processing.
The other advantage is that doing it this way you have minimal chance of inadvertently packaging contaminants within the particle. If you assemble the particle in vivo, then you quite often get exogenous DNA encapsulated within that particle and of course that is an almost impossible separation challenge, because you have two particles that appear the same from the outside and maybe have only slight differences in density.
However, the challenge then is that you really need to understand and hence control that viral assembly process, and that is really the scientific challenge presented to us.
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Today I am going to very quickly skip through some results that we have developed in terms of trying to assess of VLP self-assembly processing. To do this we have chosen Polyomavirus, which is closely related to the HPV VLP.
There were three questions to answer when addressing the key question of what is the process potential of this new vaccine class. Firstly, could we produce the structural protein in microbial cell factories, in other words in E. coli? If so, it is then very easy to produce it at very large-scale. Can we efficiently purify the precursors? And then can we use that material scientifically to understand the process of assembly, and perhaps technologically to drive assembly in vitro? And that set of technological capabilities has underpinned our question: how does a virus self-assemble?
I will show you some results where we compare in vivo and in vitro VLP self-assembly, and speak a little bit at the end as to how we might – I stress the word ‘might’, it is highly prospective – tailor these particles for influenza.
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Our approach to expression in E. coli is really pretty stock-standard: take a pGEX vector with a GST fusion tag, fuse to that the VP1 structural protein from the VLP shell, with a thrombin cleavage site so you can separate the fusion protein and the structural protein, and then stick it in E. coli and see what happens. We actually obtained this vector from a collaborator at the University of Colorado, Robert Garcea, who has done some superb work in in vitro assembly of VLPs and, indeed, expression of the proteins.
Through very simple factorial optimisation – nothing particularly sophisticated – we managed to get a 10- to 20-fold increase in the amount of soluble protein expressed. That number there, 15–20 mg/L.OD, if you put it into bioreactors, equates to maybe two to three grams per litre of soluble protein – soluble, viral, structural protein. We have just recently published that result.
We also tried codon optimisation on the vector and a whole lot of more sophisticated things, and it turned out in the end that simple manipulation of the culture conditions was the most effective way. We now may loop back and try to understand that from a metabolic perspective, using some modern gene array techniques and so forth, as to exactly why the set of conditions we hit upon had such a positive effect.
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We then used off-the-shelf technology for GST purification, basically to capture the protein. I don’t particularly want to explain this, for those of you who have not seen a purification diagram before, but this long section of the trace is all the junk in the E. coli cell lysate that is flowing through the detectors and being thrown out to waste, and to the right of that is the trace for the protein we are interested in. We have expressed a large number of variants of this protein now, displaying different antigens, and that is pretty much the trace you get for all of them. So you can change the antigen you are presenting without having to change your purification modality.
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Then we take the thrombin enzyme to cleave off the fusion protein from the GST carrier protein, and we end up with capsomeres, or building blocks, of the VP1 protein that are stable in buffer at about 20 g per litre, which is a nice starting point then to assemble a virus, and it is greater than 95 per cent purity.
Shown here is a gel-like image of an electrophoretic chip result for protein sizing, where we are showing the fusion protein and then, after cleavage, the structural protein (in this case with an inserted antigen) and the GST tag protein – so quite efficient separation of those two.
The question we then had was: have we expressed the single viral protein VP1 by itself, or is there indeed a structural element for a virus here? If one looks at the VP1 protein, one sees that it is heavily beta-sheet barrel orientated, and it should not be stable as a single protein. Indeed, it should aggregate out of solution very quickly. It turns out that in fact it is expressed as a pentamer, and so one ends up with the capsomere itself. We could observe that using static light scattering results.
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This is a size-exclusion chromatography column on an S-200 matrix, for those of you who are familiar with the technology. That is a small amount of aggregate in the mix. The peak occurring just after 20 minutes is the capsomere peak; the molecular mass of which is about 230 kilodaltons, and shown at the 30-minute mark you see the fusion protein, a dimer of GST. So that peak corresponds essentially to the capsomeric building block of the virus, shown at the top right of the slide. What we have there is five proteins in different colours, projecting interaction arms out from that capsomere. Those interaction arms I will come back to in a moment, in terms of how a virus then assembles itself from that basic building block.
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After you do the right things to the environmental conditions – and physicochemically that sounds very easy but you have to find those right conditions, it is not quite as trivial as it sounds – you can drive the assembly of those capsomeres into very nice-looking virus-like particles which are presenting the protein in a viral type of architecture. You see here an EM showing that the particles are of order 40–50 nm, which is exactly where they should be.
I was absolutely horrified by this image when I first saw it, because when I asked a student, ‘Is that good or bad?’ (we hadn’t really worked a lot with virus-like particles) the answer was, ‘Well, we don’t know.’ That was because EM is so qualitative. You take the virus-like particles, you dry them down, you have a look. What does that mean quantitatively? If I want to actually optimise this process, I need to have a number that tells me a yield, a size distribution that tells me whether it is good or bad. So we needed better analyticals.
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We found a system called field-flow fractionation. It is essentially a very sophisticated particle separation technique that works by flow method, but it gives very fine resolution of particles. What we are showing here is a size distribution obtained by that. The red line is the UV absorbance. So here is the size distribution, with a little bit of shouldering at peak 2 and a little bit of aggregate further to the right at peak 3, which of course is amplified on the blue light-scattering signal, but by and large quite a good size distribution, centred around the right size, 30–46 nm, with some shouldering.
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If we take material out of that peak and look at it under EM, we see that we get quite good-looking virus-like particles from the in vitro assembly method. What that means is that we now have an analytical method that is very quantitative, enabling us to further optimise the process and look at how much aggregate we get under different conditions.
I should say that we have now benchmarked that technique against an orthogonal whole-virus mass-spec technique that we developed in collaboration with NIST (National Institute of Standards and Technology) in the US, because they are struggling to develop assays with which to regulate the US biopharmaceutical industry. The two assays came out with essentially the same measurements, so we are quite pleased with that outcome.
So we now have good quantitative methods for looking at viruses and virus-like particles from a process perspective.
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That led us then to say that we could now make enough protein to actually do something useful with. (I trained as a bucket chemist, so I like to have a bucketful of protein before I do too much.) We said, ‘Well, what’s then driving the assembly of these viruses?’ – and therefore how could we better control it?
You might remember that I showed you an image with arms projecting out as you looked down onto the capsomere surface. Well, those arms tie in with other capsomeres and create basically a mesh-like structure that then becomes the virus-like particle. But it is a little bit more subtle than that.
It turns out that there are intrapentameric disulphide bonds here. They are in a dynamic equilibrium state, so they can associate irreversibly with other disulphide bonds in capsomeres, hence leading to aggregates if you don’t have proper redox control on the system. But more interesting is the role of the metallochemistry here, and, in particular, calcium binding on the VP1 protein. It turns out that calcium bridges have a critical role in the assembly, and we are starting to understand what that might be.
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In fact, we have developed some quite nice assays that let us thermodynamically probe the fidelity and efficiency of self-assembly. (I will explain this figure in a moment.) What we do is to take the capsomeres and put them into different chemical environments. We watch what happens to the thermodynamics of attraction between the different capsomeres. And indeed people have done this with protein crystallography by measuring something called the second virial coefficient. Essentially, if you have a moderately strong attraction, a moderately strong second virial coefficient, you can potentially obtain a crystal. It is not said that you will, but you can, or you may. However, if the thermodynamics of attraction are too strong, then you will probably obtain an aggregate. And of course if the thermodynamics of attraction are repulsive, then you will essentially have only a protein solution present. So sometimes you would like a solution, sometimes you would like a virus particle, and usually you want to avoid the aggregate.
We developed an assay, and this is the first time this has ever been reported – we have certainly not seen anything in any dynamic system where people have measured a second virial coefficient, because it is an equilibrium measure and we inherently have an irreversible process going on, so we have had to adapt the technique and the theory to be able to probe this system. What we are doing here is presenting two traces. The first one is similar to the trace I showed you a moment ago from the light scattering. In other words, it is capsomeres that we are putting down a size exclusion column, and we are capturing the peak as it comes off that column. (That is the red-line peak at the left.) And by some fairly sophisticated light-scattering techniques we are determining what the second virial coefficient is, within that peak. In other words, we are actually measuring the attraction between the particles as they are eluting from a column.
Then on the black line what we do is to change the buffer conditions slightly, and in particular put in some calcium. What we see is an immediate increase in size of the particles, but we have managed to capture the assembly process in transition, such that there are still enough capsomeres in that capsomere peak that we can access the second virial coefficient.
The two numbers we get out are quite interesting. When we are in capsomere-stable buffer (negative means attractive) we have a number that would lead people to say, ‘That should be aggregating,’ if one understands the crystallography data. And yet it doesn’t; it’s stable. So there is an anomaly there. However, when we put the calcium in, we get something that is two to three times more attractive. And then we get assembly occurring, and indeed we get VLPs out of the system.
So what has happened is that we have gone from a highly attractive system to something that is much more attractive, and then we have created a VLP. We don’t fully understand this.
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What we suspect is happening is that as we bring in the calcium, we actually fold these arms in, such that the steric barrier that is created by the arms is reduced. And indeed with some other methods – I am not presenting the data – we can detect a size reduction in the capsomere on the addition of the calcium. So they seem to fold in.
Simultaneously the calcium will alter the charge balance on that surface, and hence the electrostatic barriers to self-association are decreased. Consequently it looks as if the assembly can proceed.
So it is a fine balance of DLVO (Derjaguin-Landau-Verwey-Overbeek) type actions, which are the electrostatics, coupled with a triggered change in the steric barrier around the capsomere.
As I said, we have only just developed these tools; this is early data. It seems that if we get the conditions wrong we do indeed make it even more attractive and then we get aggregates. So it is starting to tie together quite nicely for us.
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I am going to shift gear slightly now, back onto the technological. We decided, ‘Okay, we have made these VLPs. On the old question of whether it is good, whether it is bad, at least we can measure it now. Let’s now make the VLPs themselves inside insect cells, baculovirus cells.’ And so we did that.
Shown here is a caesium chloride separation of the VLPs that we have made. It is well known that the VLPs come essentially in two bands on these systems, where the lower band is the one packaged with the contaminant DNA from in vivo assembly. So we take the top band and we assay that to benchmark against the in vitro method.
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Here you see the sort of EM image we get. We do indeed get VLPs. We also get aggregates, we also get strange structures occurring – I am not sure why. Some of those are baculoviruses, others are actually VP1 protein tubes. (It has also been shown that if you get the conditions wrong in vitro you can indeed make tubes instead of virus-like particles.)
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If we run the size distributions – this is the field-flow fractionation system – we see quite a tight distribution on the assembled VLPs from E. coli and a broader distribution from insect cells. That may relate to the fidelity of purification we can achieve on these systems.
Now I am going to shift gears for the last five or 10 minutes to talk about tailoring for influenza.
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I took this quote out of the Australian Management Plan for Pandemic Influenza. When I first read this, it struck me that this is a problem of manufacturing speed, not efficacy.
Of course, we have to have efficacy (that is a given) but assuming we have efficacy, through an appropriately reassorted virus, we then have the manufacturing problem to deal with.
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This back-of-the-envelope analysis of where we are at says that a three-month response to the first, limited, vaccination is unacceptable. And it would be desirable, in terms of scale, to vaccinate the whole population, not just the at-risk population or indeed the emergency services.
In terms of safety, making large quantities of virus as a process starting point is difficult – the technology transfer challenge, if you like – in that you require highly contained manufacturing infrastructure and a guaranteed supply of raw materials; it is potentially dangerous, because you require product inactivation; and it is generally impractical in South East Asia for the complex technologies such as cell culture, except in some instances.
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An alternative way of thinking about this – and I don’t claim to have the answer, I am more just asking questions here of this audience – is perhaps to take this capsomere, which has known surface insertion loops in it, and graft in appropriately antigenic peptides, and in essence take a VLP and change the surface of it to present, in a viral-like architecture, the antigen that is of interest.
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If you did that, you would end up with a radically different vaccine process. You would have a bacterial fermentation, a cell disruption step, perhaps an expanded bed chromatography capture system so you didn’t have to clarify your bacterial lysate, followed by a tag removal step of some description, some sort of purification step – these are all known techniques now, although you might change them, improve upon them – and sterile filtration. Now, that is interesting in a vaccine process, because essentially up to that point you only have protein, so you can sterile filter it. And then you can assemble it, package it and administer it. So, it is a radically different process.
If one uses directly the techniques I have just outlined to you, which work rather well for our VLP, then the fermenter would be a 50-litre fermenter. The whole thing would fit on a skid that would fit in the back of a Hercules, and you could dump it somewhere and using bacteria it would turn out half a million doses a week, based on the sorts of yields we are getting.
That is the sort of approach we take with water challenges in response to tsunamis. We don’t keep shipping bottles of water, we shift a reverse osmosis unit.
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This is the worst example we have, where we took a certain peptide out of the haemagglutinin protein – actually the loop A antigenic sequence from H5N1 – grafted it into our VLP and used exactly the same process. This is not a particularly good EM image; the size distributions look better. But indeed we do make VLPs with that.
So this is an example where we had not changed the process at all from the one for the wild type, yet we are able now to make a VLP that presents quite a different surface to the immune system.
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So what about the immune response? I am sure you are all sitting there asking that question. The simple answer is that I don’t have the answer yet. We have designs now ready for animal trials; we are trying to design for efficacy and we are doing a lot of thinking about that.
As we learn about the efficacy, because we are in bacteria and we are using all the tools of microbial cell factories, we can generate a new vaccine design within a week. In that context, our biological tests and not our bioprocessing become the rate-limiting step.
Also, we can do ‘ignorant’ vaccine design. If we know there are hypervariable regions in a given virus, we can simply take the template DNA, PCR amplify it, insert it straight into our vectors, and naïvely create a multiplicity of capsomeres that we can assemble into a multiplicity of VLP structures.
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To step up from our specific technology and ask what might be possible with cell-based approaches to vaccines: in terms of speed, you could insert the antigen and then use the same process, potentially. For microbial systems that means you could go from DNA to purified vaccine, potentially, in less than a week. (Because it is robust technology it could be automated at some level.)
You can use widely available, scaleable process methods, and essentially move to a dose-surplus rather than a dose-sparing paradigm. You are no longer then doing technology transfer – potentially you are doing facility transfer. You have miniaturised it down; you stick it in an aircraft. We even have some very large cargo planes in Australia now, I understand.
In terms of safety, you don’t ever make a virus, you make the protein and therefore you can sterile filter to remove exogenous virus.
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Here are some specific conclusions related to our work.
The platform seems to work well. We get quite stable, highly pure capsomeres, with a simple process and efficient assembly.
We have developed a whole raft of new characterisation tools that really let us ask the process questions, and indeed the scientific ones, as to what we are dealing with.
And, of course, as with any research we ended up with more questions than answers. In particular, some insertions are stable and some aren’t, and we are trying to understand that interaction with the VP1 protein, as to what drives that. It is actually an interaction also, as I said earlier, with the physical environment that we are dealing with.
What factors control or influence assembly? I gave you some insight into our early results on that. We are learning something new every day about how these virus-like particles come together and the thermodynamics of attraction, and the impact of physicochemical conditions.
How do we best adapt the biomolecule without affecting the bioactivity? And then how does that interact with the process? They are underlying questions for a lot of this work. Indeed, can this philosophy be brought to bear on other product classes, as our scientific understanding of this technology improves?
If I take all of that together, I end up with the question: can we design efficacy for given processing routes? The first time I wrote that question down, I came to the conclusion that this would be an impossible undertaking. And then I stood back and said, ‘Well, what have people achieved, actually, with monoclonal antibodies?’ We are doing exactly the same already. We have defined cell-culture and purification methods, and yet we can target the molecules at different antigens in different targets. So there is precedent there within the biotech industry that that can potentially be achieved.
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Of course, none of us works alone, and I have the privilege of working with a very good group at the AIBN, in terms both of colleagues and of people within my own group. Listed here is a collection of my students and colleagues. I would like particularly to thank Ian Barr, who sent us the DNA that we then PCR’d up and put into the VLPs, and also the CMM group at the University of Queensland, who taught us how to do EM.
Discussion
Question: I have a chemistry question. Theoretically the size of the self-assembled particles should be very homogeneous. Based on your estimates, there is still substantial variation. Could this be due to some of the one-time aggregate formation?
Anton Middelberg: The natural virus is not as homogeneous as we think it might be. I think that is generally true for virus. I suspect what is happening is that the physical variability in the virus also helps generate randomness that the immune system then cannot shut down. Actually, if you took virus made in the cell you would also have a size distribution rather than a perfect size. Indeed, some of the capsomeres have five-around-one and six-around-one symmetry, so there is variability in there from a chemical point of view.
Question: Does that influence the immunogenicity?
Anton Middelberg: The simple answer is that we don’t know. In fact, there is an even broader question that underlies yours: what is the interaction of the physical structure of that antigen with the immune system? People don’t understand that question. There have been immunological studies with the capsomeres which suggest that they are as efficacious as the virus-like particles, but I believe that they are actually flawed because they didn’t understand what they were immunising with.
Question: In a sense, one is struck always by the elegance of the improvements and the advances that one can make by tweaking the system – the expression, the optimisation of the assembly and so on. But considering the conservative nature of vaccine companies, and the fact that the product really is the process that got it there, the ability to use these approaches with pre-existing vaccines is going to be, I think, extremely difficult. I don’t know what experience you might like to share with us in your attempt to get the vaccine industry to adopt a new approach to the production of an existing vaccine, whether you have ever been able to achieve that, and therefore the issue of efficacy is critically important. You might say that it is as efficacious, or you think it is as efficacious as the present processes…
Anton Middelberg: No, I didn’t say that, actually.
Question (cont.): I know, but ultimately one has to demonstrate it.
Anton Middelberg: Absolutely.
Question (cont.): And therefore probably the only way to get adoption of this is in fact to select a new vaccine that can apply these principles and approaches, and then actually establish that it does work for a particular new vaccine. I just wonder what your experience…
Anton Middelberg: Absolutely. I am not saying that the efficient manufacturing of an ineffective vaccine is something that anyone would ever want to attempt to do. There are several layers at which I can answer your question. To deal with your last one first: I think of it very much as a bioprocess overlap. Think of a Venn diagram where you have a circle of things that are very easy to make, and a circle of things that are efficacious. I think what we are always doing is starting in the efficacy circle, and then addressing the bioprocessing circle. And in fact we are often then struggling to find a bioprocessing route which is robust and transferable across different sites.
So I guess what I am saying is that because of my background I am coming from the bioprocessing circle, and we are now starting to look for that overlap with the efficacy circle. And I think there are modern tools we can deploy onto that problem to then try and find an antigen that works.
Perhaps influenza is not the right disease, but I really enjoyed a couple of the talks today where people were putting up scans of peptide antigens, for example, that are efficacious – or believed to be efficacious. So there are other tools that can be brought to bear, to find antigens that are efficacious to be put into various platforms that then can be easily manufactured, independent of the particular sequence that you are putting in.
So I think there is something of a decoupling there.
Now in terms of your first question, on uptake: fortunately, I have been funded by the ARC and the NHMRC very generously and I have not had to worry about getting uptake. My simple and somewhat offhand comment to you would be that yes, vaccine companies are extremely conservative; that is why we have venture capital groups.
Question: On the peptide note, my first question is: how many peptides can you fit, and what is the maximum size? I know that there could be stability issues, depending on which peptide you use.
I don’t know if you have an answer to this second question. Do you see vector-related immune response? You might actually immunise with this vector but get an immune response to the vector and then clear the antigen. Is that going to be a problem?
Anton Middelberg: They are all very good points. To deal with the first question, concerning the size of the insert: we haven’t pushed it much above 40 amino acids at the moment. We haven’t had a need to, basically. And part of the reason for that is quite simply that, because it is in bacteria, we can run in parallel many different constructs, and we can therefore create very efficiently many different VLPs which can then be mixed together at the formulation stage – or indeed mixed together at the capsomere stage to create a VLP which itself displays a multiplicity of peptides. So we have considerable process flexibility there.
Your other question concerned the specific elimination of the carrier particle by the immune system. I think that is a real concern. There are strategies for dealing with that. Firstly, it is known that if you insert into some of these loops you can remove the background ability to bind antibodies. So if you immunise with a wildtype virus and raise antibodies to that, you can knock out the binding potential by putting the right sorts of inserts in. You can carry that a step further through pegylation, to further block out that background antibody binding effect. What then happens through the cell processing routes, I don’t know.Visit these websites
The Sir Mark Oliphant Conference Series is supported by the Australian Government under the International Science Linkages Program.
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