Fiskevaksiner og genteknologi â Internseminar - Bioteknologinemnda

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Bioteknologinemnda: Internseminar 5. september 2001other molecules are already there. How does itwork? If you look at a traditional killed or subunitvaccine, what you get is basically something calledinternalisation: When you have a typical cell withnucleus and endoplasmic reticulum, the cell takesinto itself the material of the killed bacteria, or of thesubunit components, i.e., ”dead” biological material.It’s digested by lysosomal processes into fragments.You then get the development of an immunologicalresponse based on this lysosomal system, in whichthe fragments are presented on the cell surface orexported, where it produces what is called a MHCII response, production of antibodies. It’s a specificresponse for doing that type of function.When one looks at live viruses, whether livepathogens, attenuated viruses, or DNA vaccines, themechanism is somewhat different. DNA vaccinesstimulate another part of the immunologicalresponse, where you get a vaccine plasmid insidethe nucleus of the cell. It produces a protein in thecell, which is then processed in the endoplasmicreticulum and transported to the cell surface, whereit is then presented as a MHC I system (as opposed totraditional vaccines, where you have MHC II), whichis a somewhat different immunological response. Itwould be the same whether it is a virus or whetherit is a DNA vaccine - when the virus infects the cell,the cell makes the viral proteins and that produces animmunological response.Administration of a DNA vaccineIn non-fish - cows, sheep, chickens, humans – theadministration is intramuscular (into the muscle),subcutaneous (under the skin), or in the mucus ornasal (nose spray, into the mouth). In fish, you alsohave intramuscular administration, intraperitoneal(into the peritoneal cavity) – that’s how one vaccinatesfish today: vaccinations today in fish goes into theperitoneal cavity, so the vaccine just washes aroundamongst the organs and gets absorbed from there– and immersion: you put the fish in a solution ofDNA vaccine where the fist would take the DNA up,generally through the gills, and of course swallowsome as well.Anatomy and production of a DNA vaccinevectorWhat does it look like? You have a circle of DNA.On that you have elements that one needs. The firstelement is what is called a replication origin, and thatis needed to allow the plasmid to grow in E. coli. Tomake a lot of this vaccine, you have to be able to growit and harvest it and the easiest place to grow it is inE. coli. So this element allows the plasmid to divideand replicate in an E. coli. The replication origin isspecific for bacteria, in fact only for a certain group ofbacteria. So, for example, that replication origin willnot allow it to grow in a eukaryote, for example a fish.Next, you have a gene for selection in E. coli. Generally,what is used today for experimental vaccines and inthe earliest stages of research, are antibiotic markers,antibiotics: ampicilin, kanamycin. Then you have thegene of interest, the gene that is going to produce theprotein that will give the immunological response.And you have two controlling elements: a promoterwhich controls gene expression when it is in the cells,and another regulatory sequence at the end calledpolyA, which has to do with the stability of geneexpression. Both of these are eukaryotic specific, sothat in bacteria these genes will not function. So youhave basically a system where, in bacteria, you can getreplicates of the plasmid, you can select for it and youcan grow up large amounts and harvest it, but the genedoesn’t work. When you stick it into eukaryotes, the8
Fiskevaksiner og genteknologigene will produce, but the plasmid can’t replicate.How is it produced? You have your gene, you stickit on your plasmid, you put it into bacteria, generallyE. coli, you grow it up, and then you purify it so it’s justa pure plasmid, clean of all other contaminants. Thenyou take it out and you vaccinate the fish with it.Results of DNA vaccinesI’m not going to show you a lot of results, but I amjust going to show you one example that shows youthe type of good results you can get. I could also showyou a lot of bad results, because as I said, this is nota magic bullet, this is a technique just like all othertechniques in vaccine development and everythingelse. Sometimes, with some organisms it works, orwith some genes it works, for some it does not. It takesa lot of time, and it is not a magic bullet.Typical good results (see figure): This is simplya little bit of data that shows intramuscular vs.intraperitoneal vaccination. The Y-axis shows howmany fish have died in the challenge. In the first threecases, you have vaccinated the fish with increasingamounts of DNA vaccine, in this case into the muscle;the fourth case is a control plasmid without your geneof interest injected into the muscle, where you get50% dying, whereas with a DNA vaccine you havevery few dying. In the fifth case, you have inactivatedvirus injected into the muscle. Normally in a vaccineyou do not inject inactivated virus into the muscle, youformulate it in a proper formulation and it goes intothe cavity here. So this just shows you what happenswhen you have an inactivated virus: again, it hasvery little protection. And the sixth case is if you justtake naked DNA and put it into the peritoneal cavity,again without formulation, without oils, withoutanything else: you get some protection, but not asgood as by putting it into the muscle. And this is quitecommon, both in fish and in mammals, that one ofthe best methods for delivering vaccines today is stillintramuscular.Unique features of DNA vaccines- No risk for infection, there is nothing alive.- You raise antibodies or an immunological responseagainst the native forms of the proteins.- You raise T-cell response, which is again animmunological response- You can raise long lived responses; again with fish,we are talking about fish that are alive for two years,so we don’t need a vaccine that lasts 40 years, we needa vaccine that lasts 2 years.- Good stability at low and high temperatures. As apure piece of DNA, DNA is very stable, you can do alot with it and nothing happens to it.- And of course it is very easy to use a combination ofvaccines for a combination of proteins.Limitations of DNA vaccinesI have talked about a gene of interest. Basically, themajor limitation to DNA vaccines is that it is limitedto proteins. Often the best immunological responseagainst a pathogenic bacterium is in fact raised byvery complex molecules, for example a protein thathas been modified and has extra sugars and all sortsof different modifications put on it. In the pathogenicbacteria, there are many gene products responsiblefor modifying the components.These compleximmunogenic molecules can be easy to isolate; eitheryou can use a whole bacteria, or isolate componentsof the cell wall, for example, and that componentwould give a very nice immunological response. But toprovide the cell with the same complex immunogenicmolecule, in the form of a DNA vaccine, you wouldhave to stick a huge number of genes into the animalcell and you would have to hope that the animal cellgene expression machinery will work properly withall of these bacterial genes, turn them on and off at theright times and at the right levels, so that the animalcell will actually make the compound you are lookingfor. This would be a tremendously complex problem,scientifically, and probably will never be possible.So the major limitation for DNA vaccines, is thatthey are limited to a protein. If the major or the onlyimmunological response is raised against something9
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Fiskevaksiner og genteknologi â Internseminar - Bioteknologinemnda