Advised by Shailaja Rabindron
Aug 16, 2006
During the summer of 2006, I was responsible for helping create more effective viral constructs for the production of HIV vaccine in plants.
Green plants are the lowest cost source of proteins. Additionally, proteins made in green plants are free of animal pathogens that are more likely to have the potential to infect humans. This platform is often called Molecular Farming, and it can be used to create new vaccines, make low-cost industrial enzymes, or manufacture therapudic vaccines such as monoclonal antibodies, cytokines, lymphokines, or peptide hormones.
There are two major routes for achieving Molecular Farming:
1) Permanently modifying the plant genome, thus creating a transgenic plant
2) Transient gene expression, in which RNA vectors are introduced to modify a plant's protein synthesis in such a way that it is not inherited.
My research specifically involved innoculating a transgenic variety of tobacco, modified to naturally express Alfalfa Mosaic Virus (ALMV) RNA 1 and RNA 2. I infected these P12 transgenic tobacco plants with a recombinant form of ALMV CP that contained antigenic sequences from several different viral antibody claves from the Human Immunodeficiency Virus. The aim of this study was to determine the viral yield of such a production method, and if necessary find ways to increase that yield.
While some of these claves did not significantly slow the production of viral particles in a comparison with the wild type virus, many severly affected the growth rate of the plants. Effects from minor growth stunting to complete necrosis were observed.
In response to these setbacks, it was decided to modify the recombinant ALMV CP to more closely resemble the wildtype virus in characteristics such as hydrophobicity and electronegativity. To that end, several new recombinant viral particles had to be generated, sequenced, grown, transcribed, and injected into P12 plants for testing purposes. I was able to test three separate rounds of these new viral particles and for the most part found generally negative results while modifying sequences up or down stream of the antigenic HIV sequence.
Interestingly, a single modification of one amino acid selected at random from within the antigenic sequence had a profound effect on the plants, resulting in the highest viral yield plants outside of the wild type infections. This result, while interesting, is marred by the severe decrease in immutagenetic effectiveness of the modified sequence. This area certainly deserves more attention.
The end result of the project was positive as both electronegativity and hydrophobicity reductions allowed for higher viral yields. However, the levels of production were still not nearly close enough to the wild type virus to be cause for celebration.