Friday, 16 January 2009

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.

Potato plantsPicture of potato plants from

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.

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