An edible vaccine for Hepatitis B - part 3

Part 3 - 2008 and into the future

If you're keen you could go back to read part 1 and part 2. They describe progress on vaccines against Hepatitis B virus from genetic modification of yeast to the production of vaccine in edible plants like potato, lettuce and tomato.

The technique for genetic modification I've described so far produces 'stably transformed' plants. This is enabled by the wonderful ability of the plant kingdom to grow a whole new plant out of a mature cell of an adult plant instead of doing the pollen-egg-seed sexual fertilisation thing.

The fragment of virus DNA (the antigen) plus its promoters and targeting sequences that you've constructed are packaged up and fired into plant cells. You can then pick out just those plant cells that have successfully taken up the new DNA and integrated it into their own, and grow new plants that incorporate your gene for virus antigen in every cell, including the pollen and egg cells. So your transformed plants produce a new generation of transformed plants of their own, however you choose to propagate, by cuttings or transferring pollen or whatever.

There's another way to get your carefully constructed DNA into a plant, and that's by using plant viruses. The way that viruses work is that they hijack a host's cells in order to reproduce and spread, so if you put your Hepatitis antigen DNA into a plant virus and then infect a plant with it, the plant produces your desired protein as well as propagating the virus. It's called 'transient transformation' because unless the virus gets to every cell in the plant (which would probably kill it), the viral DNA plus your new DNA isn't incorporated into the whole plant and isn't passed on to the next generation.

The advantage of this transient transformation technique using plant viruses is that you get lots of protein very quickly. To start with, they found that the levels dropped off very rapidly, as if the plant had worked out that something was going on and mobilised defences against it. This leads us to an area of genetic research called 'gene silencing' and a whole new topic that I won't go into here (and in the end, I left it out of my critique too). A way to combat this gene silencing effect was found, and let's leave it at that.

Most of this viral transformation work has been done with non-food plants, like our friend tobacco. One study, though, used a virus that can infect edible plants like cucumber, and perhaps we'll be able to eat the virus-infected plant to get immunised (the study hasn't reached that conclusion yet). Another used a fancy new viral transfection technique, admittedly on tobacco leaves again, but managed to produce extraordinary high levels of Hepatitis B antigen in only 14 days.

The interesting thing about this last study is that it was done by the same team that published the original paper I was reviewing, when they were expressing the antigen in potato and feeding it to mice. They are still working on an edible vaccine, but I think the concentrations they are getting are so low and it takes so long to grow the different plants that this parallel line of research is quite attractive, Perhaps producing Hepatitis B antigen by this viral transformation might form a bridge between the yeast-derived vaccine and a future stably transformed plant supplying an edible vaccine.

Then there are one or two other issues that will need to be sorted out for an edible vaccine. In the paper describing this success with viral transformation, they write: "Although our original research goal was to provide inexpensive, easily administered vaccines in the form of edible transgenic plant material, it is realized that plant-derived vaccines, like any other drug, will be subject to the strict regulations of the US Food and Drug Administration or other similar national agencies. Requirements, including dose standardization, must be met before a plant-derived vaccine is approved. To ensure dosing consistency, vaccine antigen-expressing plant materials must be subjected to downstream processing to some extent, including steps ranging from simple freeze-drying to chromatography purification."

For my coursework I had to put all of this into 2000 words. Obviously I left out some of the explanation, but I had to put in quite a lot more technical detail. I read more than 30 scientific papers, and referenced 20 of them in the final version. I have absolutely no idea whether I will get high, middling or low marks for this bit of work, because it will depend on whether I've interpreted the assignment correctly and written about what they expected me to write about with the correct amount of detail and accurate referencing. I've found it thoroughly fascinating, though, and it all stands me in good stead for the exam.

The last lecture in this module was delivered by one of the team who was responsible for Dolly the cloned sheep. It's been the only lecture that touched on genetic modification of animals; all the rest has been about plants. It's been the best module of the six I've been doing this semester, and isn't even part of the core syllabus. Psychology has been very interesting too, and I've blogged a little bit about that. The modules about the effects of nutrition, exercise and hormones on metabolism have also been good.

The other two modules have only been OK. Immunology is an interesting subject but was taught very badly, and what was described as Mammalian Biochemistry turned out to be about embryology and early organ development. If you think that viral vectors, transcription factors and manipulating DNA are complicated, it's nothing compared to the complexity of complement cascades in the immune system, or the factors that affect the early development of your heart, liver, kidneys and the rest of your innards, starting with one single cell - the fertilised egg. Miraculous.

An edible vaccine for Hepatitis B - part 2

Part 2: from 2000 to the present.

You might like to read Part 1 of this story first, but no matter if you aren't that keen. I left you with an experiment to try and produce an edible Hepatitis B virus vaccine in potatoes. The principle was proved successful, but there wasn't enough antigen in the potatoes to make it a serious contender at this stage.

The first option that might strike you as sensible is to try a different plant. Not just because it might work better, but because raw potato isn't generally considered an appetising food. Apart from the lettuce experiment, different teams have done similar trials in tomatillo (a Mexican fruit), banana, rice and tomato, although I'm not sure what the rice team were thinking, given that cooking the rice would destroy most of the protein. None of them tried feeding their plants to humans, but the team that wrote 'my' paper had a go with potato.

Most, but not all of the human volunteers who ate the transgenic potato had the desired immune response, and they didn't even bother with the adjuvant this time (if you remember, the adjuvant is something to make sure the immune system doesn't ignore your vaccine). Still, none of these foods made enough antigen yet to make a viable edible vaccine.

There were two other paths still being explored, both of them involving tinkering with the transgene (the virus DNA fragment that is put into the plant) by a) choosing a different bit of the virus as the antigen, in case the bit they first chose isn't the best bit - if they could get a better immune response from another fragment, then they wouldn't need as much protein in the vaccine, or b) putting different promoters and targeting constructs in it.

The promoter is the bit of DNA that tells the plant cell machinery to "start transcribing DNA into mRNA from here". You may know that there's an awful lot of random DNA that we don't think is being used - 'junk' DNA. One way the cell knows which bits to use and which bits to leave alone because of certain sequences called promoter regions. Sometimes these promoters are permanently blocked so that the DNA is never transcribed and the protein is never made. For example, every cell in the body contains the DNA that could make the milk protein casein or the blood protein haemoglobin, but we only really want them to be made in mammary cells or bone marrow cells respectively, so it's permanently blocked everywhere else.

Of course, I'm writing as if the cell 'knows' what to do. That's one of the miracles of biology - of course it knows nothing. At molecular levels, which is where this is all going on, it's all about electrical charges and attractions between individual atoms based on where their electrons are. Molecules float about in cells, and randomly bump into other molecules, which give them a push in one direction or other in the same way that comets float about the universe getting pushed about by stars' gravitational fields. Billions of years of evolution (and magic) means that this random motion is directed into interactions that make life possible. We're all about chemistry inside our cells, and chemistry is all about physics.

Where was I? Oh yes, promoters and targeting constructs. If promoters are at the beginning of a gene saying 'start making your mRNA here', then targeting constructs are at the end of the gene, and tell the cell where to make the protein, or what to do with it after it's made. This is where you try to direct the vaccine protein to the fruit or tuber rather than the leaves or flowers, or where you could try and get it secreted, or stored in chloroplasts or the vacuole or other internal areas of the cells. This bit is quite technical, but they've tried a few different ones with variable results. The trouble is that storing a lot of foreign protein carries the risk of poisoning the plant, so there's an amazingly clever technique called inducible expression.

I've mentioned that there are promoters that are permanently blocked in some cells so that their associated gene DNA can't be transcribed. Transcription factors are molecules or complexes that do this blocking, by attaching themselves to the promoter region of DNA. It's possible to treat the plant with something that changes the configuration of the transcription factor so it stops being attached to the promoter. That means that a gene that previously was not transcribed becomes unblocked, and can start making mRNA and then protein. The thing that can change the transcription factor could be as simple as cold, or ethanol, or 'wounding'.

So you could grow your normal potato or tomato or whatever, harvest it normally, then chill it, expose it to ethanol fumes or pound it to a pulp. All the transcription factors would detach from the promoters in every cell, and the DNA would get busy and make your Hepatitis B antigen protein. Brilliant. This has been tried out in tobacco leaves, but not in an edible plant yet.

So we are now at the stage when we've proved the principle of the edible vaccine, but we’re struggling to get the concentration high enough in the edible part of the plant without killing it in the process. There's one other alternative, but for that you'll have to wait for Part 3.

The picture of the potatoes came from www.bbc.co.uk

An edible vaccine for Hepatitis B - part 1

[I first posted this and the following two segments in December, and then decided to take them down because some students hadn't yet reached the deadline for handing in their work.]

I finished the Molecular Pharming coursework at 8.30 p.m. on Thursday, for handing in on Friday. That was a tough assignment, but so very interesting. It also brought together most of the topics that we've covered in this module, and emphasised how very cutting edge this course has been.

Picture of potato plants from http://www.veggieharvest.com/

The research paper I chose to write about was published in 2000, and covered a study on producing a vaccine for Hepatitis B in potato tubers.* We had to produce a critique of the paper, discussing the findings and its context, including what has happened since that time.

To create a vaccine, you have to find something (an 'antigen') that is so similar to the disease that the body responds by creating antibodies and immune memory cells specific to that antigen. That way, if the real thing turns up later, the immune system is ready to fight straight away and the disease doesn't have a chance to gain a foothold. In the most famous case, a cowpox antigen was similar enough to prime the immune system against smallpox. Sometimes they use dead or weakened bacteria, and in the case of Hepatitis B they found a bit of the virus 'envelope', its outer coat, that did the trick.

The next step came about through the new science of genetic manipulation. Normally DNA makes mRNA which makes protein. In this case, they took the bit of virus coat protein and reverse engineered a bit of DNA, then inserted it into the genome of a species of yeast. Through what can only be described as magic, the yeast produced a bit of protein that was identical to this bit of the virus. If the yeast is allowed to get on with living in a huge vat, eventually they can extract the protein made by the yeast (Hepatitis B virus antigen), purify it, process it, and create a vaccine by mixing it with an 'adjuvant': something to wave a flag at the immune system to say "Oi - look over here - I'm a disease!"

I think (without looking at my notes) that a commercial vaccine was first produced in this way in 1981 or 1982. [Aside: I find it fascinating that this was well within my lifetime, but before nearly all of my fellow students were born.] It's very effective as a vaccine, but there are disadvantages: it's expensive to make, needs to be kept sterile and refrigerated, and must be administered by injection on three separate occasions. In the places where Hepatitis B is most prevalent, it's not affordable, sterile refrigerated conditions are rare and getting patients to conform to the three injection regime is difficult.

What I've learned this term about genetic modification, and the ability to manipulate bits of DNA (genes) is staggering. The lecturers talk blithely about creating desired sequences, knocking out genes, detecting single nucleotide substitutions, and even targeting the translation of mRNA into specific locations as if it were the most normal thing in the world. You want to make a protein in fruit but not in leaves? Easy, stick a fruit-specific stretch of DNA on the front of the gene. You want your protein secreted out of the cell once it's made? No problem, stick a secretory signal peptide on the end of the DNA. Want it? You got it. Unbelievable.

Using 'simple' organisms like yeast and bacteria to make useful proteins for us seems pretty routine. Insulin which used to be extracted from pigs can now be made in human-identical form without any input from the animal kingdom. To me, that would seem to reduce animal-based research (unless you consider bacteria or yeast to be animals), but it's genetic modification, because you've modified the genes of the bacteria and the yeast. And some people are simply against genetic modification. If I've learned anything, I've learned that every case is different. Some GM techniques benefit humanity. Some GM techniques benefit large global corporations and their shareholders. Some GM techniques take us close to ethical boundaries, if not across them. But it all needs to be paid for, and nobody can afford to spend a lot of time at the cutting edge without drawing a salary of some kind.

Anyway, I digress. I left you with an effective Hepatitis B vaccine produced by yeast, albeit with drawbacks. Meanwhile, things had been moving in the world of plant science. If a yeast could be genetically modified, why not a plant? Turns out, it can, and what's more, the antigen produced by the plant is better than the yeast, because it needs much less processing afterwards - plants assemble it into a form that resembles a virus much more closely than the yeast can manage. They started doing experiments with tobacco plants (don't ask me why tobacco), and extracted enough protein from leaves to show that an immune response could be established by injecting it into mice.

Obviously there's still a processing overhead when you have to extract your product from tobacco leaves. The obvious next step was to try this out in edible leaves. In 1999 there was a small scale trial where three people ate transgenic lettuce together with the adjuvant (the stuff that makes sure the immune system is paying attention), and bingo! their immune systems recognised the virus. Success. Well, actually, only two out of three showed a response, but still.

Meanwhile, the team that wrote 'my' paper were hard at work making a potato to do the job. First they replicated the mouse experiment and showed that their transgenic potato provoked an immune response when the mice simply ate the tuber with adjuvant - no purification from leaves or injection needed. The main disadvantage of eating the vaccine is that you need a lot more of the antigen than you do when it's injected in a pure form, partly because you digest some of it, and partly because the immune cells in your gut are a bit less sensitive, otherwise they might respond wrongly to everything you eat. And plants only make teeny tiny amounts. You'd have to eat a whole lot of raw potato (or lettuce).

So they tinkered with the DNA to try and make the potato express more protein, and did quite well, increasing the level about forty-fold. As always, there's a catch - potato plants don't naturally produce a viral envelope protein, so when they are asked to make quite a lot of it, they get a bit sick. This is where the paper in 2000 ends: edible potato producing vaccine protein, but still not enough of it. For the next eight years' work, you'll have to wait for Part 2.

* Richter, LJ, Thanavala, Y, Arntzen, CJ & Mason, HS 2000, 'Production of hepatitis B surface antigen in transgenic plants for oral immunization', Nature Biotechnology, vol. 18, no. 11, pp. 1167-71.