Genetic modification of insects part 4

Using Mosquitoes to Conquer Disease Through Vaccination

One of the things I’ve been talking about here on Biofortified is the concept of a ‘pest’, which is a completely anthropocentric term. Different insects can be pests at one part of their life cycle and be totally cool in another. It’s one of those weird science paradoxes which make the field of entomology so much fun.

In my last series of posts I discussed a new way to bring down pest populations by letting transgenic insects mate with wildtype insects and letting their offspring wither and die from a toxin which builds up inside of them. It’s really just a variation of a technique that’s been around since the ’50s that uses a gene that codes for a toxin (part 2) in place of radiation (part 1). It’s great for everyone involved (except for the mosquitoes – part 3), and it could even lead to the technique becoming more widespread by nixing the use of radiation all together.
It turns out that on top of all that we may be able to make mosquitoes work to our advantage.
Vaccines work by stimulating an immune response. In short, vaccines are made by either injecting a weakened version of the pathogen or a part of a pathogen like an important protein that’s part of the pathogen into a person. This coaxes the body into producing proteins called ‘antibodies’ which bind to the pathogen. From here, one of two things may happen… either a white blood cell finds the antibody-coated intruder and eats it (think pac-man) or a complex forms on the surface of the intruder and makes lots of holes all over the intruder which will eventually kill it. Injecting bits of pathogens or weakened pathogens into the body essentially gives your immune system a ‘cheat-sheet’ for something it may eventually encounter.
Your body can make antibodies to pretty much any non-self protein, which is incredibly useful. When mosquitoes bite you, they inject numerous proteins in their saliva which act as local anesthetics, anti-coagulants, vasodialators, so on and so forth. You actually make antibodies to these proteins and that itching after a mosquito bite is actually an immune reaction to the saliva. I work with antibodies in my lab. I essentially look for insect proteins which allow cells to communicate by using antibodies bound to enzymes to cause a chemical reaction that I can see. These antibodies are produced by injecting a protein into an animal and then harvesting them so we can work with them.
So in short you have an immune system which creates antibodies to foreign biomolecules and uses them mark targets for destruction. You’re constantly under assault from parasitic flies which inject you with proteins to which you create antibodies. We can use antibodies raised in other animals to look for proteins by using antibodies raised to antibodies that are attached to enzymes.
If you put these two bits of information together, you begin to see how this could be a useful tool because you may potentially be able to use mosquitoes to produce and distribute vaccines. A paper was recently published in the journal Insect Molecular Biology which I think is a good start towards this goal and makes me grumble a bit near the end…  but we’ll get to that when the time comes.

Let’s first walk through the image above to explain how everything works (click to see larger image). When a gene gets turned to RNA and then protein, the first step is binding of an enzyme called ‘RNA Polymerase’ at a promoter. Promoters essentially tell RNA polymerase how much RNA to make and where to make it. Above, Yoshida et. al took a sequence that’s almost always expressed and put it close to a promoter for a protein called ‘Anopheline antiplatelet protein (AAPP)’ that is expressed in mosquito saliva. Doing this allowed them to create a protein which is always expressed, but only in the saliva.
Now for the big question… what did they put into the mosquito’s saliva?
You know how I love paradoxes? Sand flies are an awesome example of paradoxes in action. Sand flies carry Leishmania which is a horrible parasite which causes your flesh to form a giant sore and pretty much melts your flesh off. Different Leishmania species cause sores in different areas. Some cause mucocutaneous leishmaniasis which is where the victim’s face slowly gets eaten away, and there’s another form called visceral leishmaniasis which is almost always fatal. It’s a gruesome, devastating disease.
Here’s the odd part: Sand fly saliva aids in parasite transmission, but can also protect those who have been bitten. When a sand fly bites someone, something in their saliva seems to help along the infection. However, those who have been bitten by a sand fly recently are better able to fight off Leishmania infection. It’s weird, I know. Those who are vaccinated with SP-15 from sand flies are protected against Leishmania infection.
The test below the picture of the gene’s set up is a Southern blot, which is essentially the scientists looking for the DNA of the genes they inserted to make sure they got there.
There’s some other stuff on there, as well. They fused SP-15 with a red fluorescent protein to be able to see if SP-15 was being produced because production of the desired proteins are just as important as making sure the DNA actually got into the mosquito. The results, shown below, are pretty neat looking (click to see larger image).

What you’re seeing is a fluorescent red protein fused with SP-15, which allows us to see that SP-15 is being produced (along with a western blot of SP-15 to make sure nothing funky’s going on).
To look to see if they could use the mosquitoes to vaccinate mice, they fed the mosquitoes on the mice repeatedly with the mice receiving 1500 bites over the course of four days. After feeding the mosquitoes on the mice, they looked for mouse antibodies by immobilizing recombinant or synthetically produced SP-15 and then washing it with filtered mouse blood, which would let any antibodies present bind to SP-15. The mouse antibodies which bound to the SP-15 then were detected with goat antibodies raised to mouse antibodies linked to an enzyme which allowed the researchers to visualize that mouse antibodies had bound to the SP-15.

So… I think they showed that mice could potentially be vaccinated against SP-15. Their titers were low (activity at 1:300 dilutions was the highest), and the authors correctly noted this. To give you an idea of what ‘normal’ activity would be, I use 1:5,000 and 1:10,000 dilutions of antibodies for my work and that’s a bit high. Not seeing activity at a 1:300 dilution means the antibody concentrations are pretty low in that plasma.
They also didn’t actually do tests which would allow them to see if the mice actually fought the Leishmania parasites off. I also don’t like the fact they didn’t do a western blot on the mosquito saliva, but the fact the saliva was glowing red from the red fluorescent protein fused to SP-15 means that chances were pretty good the mosquito was salivating the protein. I don’t like the fact they didn’t make an effort to quantify how much SP-15 was coming out in the saliva. The fact the mice produced antibodies to SP-15 is good enough for now because it shows that the SP-15 protein was actually leaving the mosquito, but it seems like a big step to go from SP-15 in the salivary glands straight to vaccination. Minor gripe… it shows the same thing but with a lot of extra added effort. It’s not a fatal flaw, but I would have done things a bit differently.
Moving this system to humans would be another story altogether. 1500 bites over four days is about par for the course when it comes to animals. Humans, on the other hand… I’m not sure 400 bites a day would happen on most Americans. Also, if you consider the mouse’s size and the low antibody titers they got, I’m not even sure this would be sufficient to replace traditional needle delivered vaccines. I’m just not convinced that this would be useful in it’s current form. As proof of the concept, however, I think it shows potential and warrants further development. Not much more than that at this point, though.
The authors conclude with a statement I disagree with:

The concept of a ‘flying vaccinator’ transgenic mosquito is not likely to be a practicable method of disease control, because a ‘flying vaccinator’ is an unacceptable way to deliver vaccine without issues of dosage and informed consent against current vaccine programmes. These difficulties are further complicated by the issues of public acceptance to release of transgenic mosquitoes. Therefore, we intend only that the present study makes available a model system using a salivary gland-specific promoter as a potential tool to elucidate the saliva–malaria sporozoite interactions

I think they’re right in that this couldn’t be used directly for humans, but I think this could still be very useful in the elimination of diseases. I think they’re right in that there are pretty serious ethical concerns as well as practical concerns with using these guys as vaccine vectors. Gene linkage, pathogen mutation (such as what we see the flu do yearly), and and a bunch of other things like how quickly we’re able to produce new vaxquitoes could potentially render this approach untenable. More research is needed before we can even consider this as a public health tool.
Despite the ethical concerns, I still think this might have some use because not all diseases are strictly human diseases. Many have animal counterparts in their lifecycle and we could potentially use these mosquitoes to vaccinate potential reservoirs.
Let’s ignore their crappy titers for a second and assume we can get this working to the point where we could get this working on a large scale. Introduce the mosquitoes to the wild, get them feeding on non-human hosts by… say, removing the equipment that allows them to find mammalian hosts. We could potentially use this system to vaccinate a massive amount of wildlife with the intention of eliminating disease reservoirs which are important in the transmission cycles of many different diseases.
Let’s take West Nile as an example. West Nile is mainly transmittesd between birds by bird-feeding mosquitoes. It only gets into humans when a bird-feeding mosquito feeds on humans in lieu of avian bloodmeals. If we were to take mosquitoes which feed exclusively on birds, transform them with this gene and then make it so their larvae die using the last set of genes I talked about (part 2) and then release them before their hosts migrate for the winter, this would create a system which would vaccinate birds of something like West Nile without the issues of informed consent raised in humans.
If we look at this as a tool to vaccinate disease reservoirs, we could achieve the same goal of reducing the prevalence of or eliminating the disease while avoiding the ethical concerns of informed consent raised by using these guys to vaccinate people. I’d say it’s a win all around.
Being able to use mosquitoes to deliver vaccines is a pretty neat tool, but let’s face it… I can see the antivaxxers needlessly freaking out over this.
Yamamoto, D., Nagumo, H., & Yoshida, S. (2010). Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding Insect Molecular Biology, 19 (3), 391-398 DOI: 10.1111/j.1365-2583.2010.01000.x