Expression of ETEC Enterotoxin in Tomato Fruit and Development of a Prototype Transgenic Tomato for Dissemination as an Oral Vaccine in Developing Countries

The broad objective of the project was to develop a feasible approach to combat diarrheal disease caused by ETEC through the development of a low-cost oral immunogen in tomato fruit, expressed in the context of a prototype tomato that would answer the shortcomings of plant oral vaccines, especially in terms of produce handling and control of gene escape. Specifically, the goals for Boyce Thompson Institute (BTI) on this project were to develop transgenic tomato lines that express the enterotoxigenic E. coli (ETEC) heat-labile enterotoxin (LT) subunits A and/or B for use in oral edible vaccines, and to optimize expression and assembly of these antigens in tomato fruits.LT-B is a useful vaccine antigen against ETEC disease, since antibodies against LT-B can prevent binding and delivery of the holotoxinLT. Mutant forms of the toxic LT-A subunit that have reduced toxicity can be co-expressed and assembled with LT-Bpentamers to form mutant LT (mLT) complexes that could be used as mucosaladjuvants for other oral vaccines. Work on the project is continuing at Arizona State University, after Dr. Mason moved there in August 2002. A number of approaches were taken to ensure the expression of both subunits and bring about their assembly inside the transgenic fruits. Initially, expression was driven by the fruit-specific E-8 promoter for LT-B and the constitutive CaMV 35S promoter for LT-A(K63). While LT-B accumulated up to 7 µg per gram ripe fruit, assembled LT-K63 was only 1 µg per gram. Since promoter activities for the two genes likely differed in cell type and developmental stage specificity, the ratios of A and B subunits was not optimal for efficient assembly in all cells. In order to maximize the chance of assembly of mLT in fruit, we focused on constructs in which both genes are driven by the same promoter. These included co-expression plasmids using the 35S promoter for both, while switching to attenuated mLTs (LT-R72 and LT-G192) that have shown greater potential for oral adjuvanticity than the initial LT-K63, and thus are better candidates for a plant-derived adjuvant. Other, more novel approaches were then attempted, including several new vectors using the tomato fruit-specific E8 promoter driving expression of both LT-B and mutant LT-A, as well as a dicistronic construct for co-expression of both LT-B and mutant LT-A genes from a single promoter, and a geminivirusreplicon construct. We describe in the Appendix the results obtained in transgenic tomato lines transformed with these constructs. Overall, each contributed to enhanced expression levels, but the assembly itself of the holotoxin to high levels was not observed in the fruit tissues.  The Israeli lab’s specific objective was to develop transgenic tomato lines expressing the LTholotoxin antigen bearing attributes to prevent gene escape (male sterility and orange fruit color) and to improve the dissemination of the oral vaccine (long shelf-life tomato cherry fruit or tomato processing background). Breeding lines bearing a number of attributes to prevent gene escape were developed by combining material and backcrossing either to a tomato cherry background, or two different processing backgrounds. Concomitantly, (these lines can be utilized for the creation of any future oral vaccine or other therapeutic-expressing tomato, either by crosses or transformation), the lines were crossed to the holotoxin-expressing tomatoes received from the United States, and this transgenic material was also incorporated into the backcrossing programs. To date, we have finalized the preparation of the cherry tomato material, both non-transgenic (bearing all the desired attributes), and transgenic, expressing the holotoxin. The level of expression of LT-B in the cherry fruits was comparable to the original transgenic tomatoes. Since it was not higher, this would necessitate the consumption of more fruits to reach a desired dose. A final backcross has been made for both the non-transgenic and the transgenic material in the processing lines. Auxin sprays resulted in high percentages of fruit set, but the processing genotypes gave many puffed fruits.