[AIDScience] Could you tell us about this edible HIV vaccine, which I understand is an SIV construct at this point?
[John Howard] Correct. What we have been trying to do in general is to produce a number of subunit vaccines in transgenic plants and, rather than inject them, feed them orally to animals or humans. The first step is to get these proteins to express in our system and to put them into corn. There are a number of technical reasons why we want to do this, as well as some safety reasons—in terms of human safety. We have done that now for a couple of viral proteins and have figured out how to make them express well in corn. They were then fed to animals to test if we could get an immune response and also protection from challenge in those animals.

Protection being the most important purpose.
Right. These studies have been going on for a couple of years now, and, since we were able to show efficacy, we wanted to look at a couple of important human viruses. HIV is one of those. The first step along that route was to determine if this protein of HIV—this particular class of proteins—would work well in corn. Would it express at high enough concentrations? Would it actually fit the model we have developed? We have recently shown that it expresses well.

What other systems have you tested in your transgenic corn model?
We have tested several. In animals, we have tested transmissible gastroenteritis virus (TGEV) of swine (1, 3, 4). It produces an immune response and also protects pigs from challenge when given orally. Also in animals we have tested an oral vaccine in mice against the bacterium that causes traveler’s diarrhea in humans (2, 3, 4). We have shown that it works in mice and are now preparing to run human clinical trials in a month or so.

Where is gp120 expressed in the transgenic corn plant?
We express the protein in the corn seed itself for several reasons. This is the edible part of the plant and there is a high concentration of protein, which happens to be stable for a long period of time. Also, this part of the plant can be formulated into something really palatable for people to eat. It does not change the taste of corn, and we can [process] it into cornmeal, for example, or other products that people would want to eat.

Is gp120 expressed in native form in the corn seed?
Yes, it is.

What about the quantities of gp120 expressed?
We screen our transgenic plants for a while until we select the ones that have the right level of expression. We have found that when we propagate these, their level of expression is consistent and stable. More importantly, once the protein is in grain it is stable for a long period of time. We have had proteins expressed in corn grain that remained stable in the seed for up to 2 years. This is very important for vaccines because of the possibility of being able to ship them to places in Asia or Africa where refrigeration is not always possible with conventional vaccines. In addition, as mentioned, the corn can be processed into something like a wafer or tablet that people can eat.

When I first came across this story of transgenic corn expressing gp120, I figured the main reason was to make large amounts of the protein.
We can do that too. In theory, one could do it for both reasons [either to make large amounts of protein or as an edible vaccine]. Our company has a number of other pharmaceutical products that we are producing in transgenic plants, just because it is a good, cheap way to make them rather than to use them as vaccines. For gp120 we are actually targeting the edible property as a higher priority, and I am not sure at this point of all the hoops that we will have to overcome. If we wanted to produce gp120 in corn just as a source, I think everybody would accept that a lot easier. So the edible vaccine concept is going to take a little while to develop, because people are going to have to get familiar with what we are doing. But I believe that it can work, based on the results of the other two systems we have worked with. It will just be a matter of working out the conditions.

Based on your previous studies, are you able to introduce more than one antigen in the same transgenic plant, for example, expressing gp120 together with Gag?
We have not done that combination yet, and this is an obvious thing to do for this one system. We have done combinations with multiple antigens with other proteins. Without going into the mechanics of this, I can tell you that yeast or bacterial systems are really designed to reject a foreign protein. The corn seed is not. To produce a high concentration of a foreign antigen, it still turns out to be a very small percentage of the total protein in a corn seed. We can introduce multiple antigens in the same plant without any effect like loss of yield on the plant. We have put up to five genes in the same plant without running into the limits of what can be introduced into the same transgenic plant. Alternatively, different antigens could be expressed in different plants and then the cornmeal [could be blended] together. A third way is to express antigens in individual plants and then do traditional hybrid crosses and blend them through genetics rather than by recombinant DNA techniques. Corn allows that kind of sexual crossing. We have used this way as well, so we actually have quite a few options. The first protein for SIV is the first step for the AIDS vaccine project. We are really quite happy to see that it is expressing quite nicely. It has opened the door to all the other directions we want to go now.

Is the gp120 produced in transgenic corn immunogenic?
Right now, we know that the antibodies that normally recognize native gp120 recognize the gp120 expressed in corn, so in that sense it is antigenic and has no obvious differences. This work has been done in vitro, and the next step is to go in vivo and see if that will hold up. We expect it will, but we have not tested it yet.

Were the antigens you have previously tested—the B subunit of Escherichia coli and TGEV of swine—immunogenic and also protective?
That is correct. We tested their immunogenicity in vitro and also in vivo, confirmed by neutralization assays. Furthermore, the oral doses we gave the animals induced protection. In the case of TGEV, the pigs were challenged with the disease-causing live virus. In the case of the E. coli subunit, we tested the oral vaccine in mice and then challenged the animals with heat-labile endotoxin (1, 3, 4). We have another project with hepatitis B virus in transgenic plants, which is in a more advanced stage than the HIV project.

Tell me more about ProdiGene.
ProdiGene is a privately held company that has been around for about 5 years. We plan to make it a public company as soon as the market is in more favorable conditions. We are a biotech company that focuses on producing proteins in plants for pharmaceuticals or industrial markets. Edible vaccines are the big platform technology that we have. It has been one of our long-term projects, although it is now becoming more short-term as things are starting to work. We also produce pharmaceutical proteins and industrial enzymes, some proprietary and in collaboration with industrial partners.

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