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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 19, 2005 as doi:10.1096/fj.04-3215fje. |
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* Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India; and
National Research Center for Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India
3Correspondence: Center for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. E-mail: rakeshbhatnagar{at}mail.jnu.ac.in; or rakbhat01{at}yahoo.com
SPECIFIC AIMS
The goals of this work were to 1) express pagA gene coding for anthrax immunogen in an edible crop (tomato); 2) carry out in vivo studies for studying antibody response generated by protective antigen expressed in nuclear transgenic tomato plants; and 3) enhance the expression of protective antigen in plant cells in an environment friendly manner.
PRINCIPAL FINDINGS
1. Successful expression of pagA in transgenic tomato plants
Transgenic tomato plants were generated with an aim of expressing pagA gene in an edible crop. The Agrobacterium-mediated leaf disc transformation of 10- to 12-day-old cotyledonary leaves resulted in the generation of nuclear transgenic tomato plants. These transgenic plants were free of any phenotypical aberrations. The molecular analysis of genomic DNA from these plants was carried out using polymerase chain reaction and Southern hybridization. While PCR studies confirmed the presence of pagA in the genomic DNA, Southern hybridization results showed the integration of the transgene in the nuclear genome.
The total soluble protein (TSP) extracted from the nuclear transgenic tomato plants was separated by SDS-PAGE and subjected to immunoblot analysis using polyclonal antibodies raised against recombinant protective antigen. The presence of protective antigen (PA) in the TSP was ascertained by the appearance of a specific 83 kDa band. Different expression levels were detected in total soluble proteins extracted from different transgenic plants. Primary antibodies were able to detect PA at a dilution of 1:200, which is indicative of low protein expression levels. An in vitro cytoxicity assay was carried out to confirm the functional properties of the PA expressed in transgenic tomato plants. The plant expressed PA was able to lyse macrophage cells when added along with lethal factor, another component of lethal toxin. The quantitative estimation of percentage killing was recorded after addition of MTT dye and measuring the absorbance on an ELISA plate reader at 540 nm. The results showed a 20–77% killing by protein samples extracted from different plants, corroborating a differential expression profile of PA observed in immunoblot detection results.
These results established the successful expression of PA in an edible crop.
2. Protective antigen expressed in nuclear transgenic tomato plants was able to generate neutralizing antibodies
The functional protein expressed in nuclear transgenic tomato plants was checked for its ability to generate a PA-specific antibody response. The total soluble protein extracted from nuclear transgenic tomato plants was tested for the generation of PA-specific antibody response using mouse animal models. Up to 300 µg of total soluble protein along with complete Freund’s adjuvant was injected through intraperitoneal route. Three booster doses of similar amount of protein were given in the presence of Freund’s incomplete adjuvant. A negative control of animals received TSP from nontransformed plants, whereas the positive control group was administered recombinant PA. The generation of PA-specific antibody response was measured by a direct ELISA, which gave the titer values of the order of 1600. In vitro protection assay was carried out on macrophage like cell line RAW 264.7. The protection of macrophages from the action of lethal toxin (PA+LF) was observed in the presence of antisera isolated from immunized animals (Fig. 1
). This indicates the presence of neutralizing antibodies in the animals immunized with plant-expressed PA.
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These results are significant in establishing the protective nature of antibodies raised against PA expressed in nuclear transgenic tomato plants. But the generation of low antibody titers (1600) is a matter of concern and could be addressed by increasing the expression levels of PA in plants.
3. Chloroplast transformation technology can be used to address the concerns associated with the nuclear transgenic crops
The three main concerns pertaining to nuclear transgenic crops expressing PA are 1) low expression levels; 2)inconsistent expression profile among a population of transgenic plants; and 3) probable environmental hazards. We therefore attempted to harness the potential of chloroplast technology to effectively answer these concerns. Initially, pagA was cloned in a chloroplast-specific vector (pLD-Ctv) after addition of upstream regulatory elements (Fig. 2
). The expression of PA was checked in Escherichia coli owing to the conservation of plastid and bacterial transcription machineries. Biolistic process was used to transform tobacco leaf chloroplasts under optimized conditions of osmoticum composition. The bombarded explants were grown on spectinomycin containing selection media. The transplastomic samples were checked for pagA gene integration by PCR. The TSP extracted from transplastomic samples was analyzed for the presence of PA by immunoblot detection. A consistent profile of protein expression was obtained in all the transplastomic samples. Higher antibody dilutions (1:5000) were able to detect PA in the TSP, which is indicative of higher protein yields. The ELISA results estimated 
8% of PA in the TSP extracted from transplastomic samples. This is a multifold increase in the expression level of PA as compared with nuclear transgenic plants. The functional efficacy of the PA expressed in transplastomic samples was found similar to recombinant protective antigen.
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Since chloroplast inheritance is maternally regulated, there is no possibility of genetic contamination by pollen dispersal. This concept of gene containment associated with chloroplast technology has best addressed the concerns of environmentalists and provided a direction to produce pagA transgenic crops in an environment friendly manner.
CONCLUSIONS AND SIGNIFICANCE
This study demonstrates the feasibility of expressing anthrax immunogen in an edible crop followed by the validation of its ability to generate neutralizing antibodies. Biolistics was used to enhance the expression levels of PA and address associated concerns of nuclear transgenic crops.
The development of a new generation vaccine against anthrax gains prominence in the wake of the realization of the pathogenic potential of this disease. Apart from the possible threat as a biological warfare agent, anthrax epidemics occur worldwide. This disease is a cause of concern for developing nations where lack of proper healthcare may further aggravate the associated problems. The best prophylaxis for both humans and animals against anthrax is vaccination.
Several undesirable side effects associated with the currently approved vaccine have fueled research for developing a new generation vaccine. This study provides a thrust in this direction by achieving two important milestones toward generation of an edible vaccine against anthrax.
Nuclear transgenic tomato plants were generated with an aim to express anthrax protective antigen (PA) in a palatable crop. The successful expression of biologically active protective antigen in plants is encouraging. However, the differential expression profile coupled with low yields observed in a population of transgenic plants has highlighted the roadblocks that may come in the way of developing uniform and effective regimen for the vaccines. The ability of plant-expressed PA to generate neutralizing antibody titers gives a promising stand to the plant-expressed PA. These antibodies conferred protection to the macrophage cells against the action of anthrax lethal toxin as evidenced by in vitro protection assay. But the generation of low antibody titers, which is probably the consequence of low expression levels, remains a cause of concern in using nuclear transgenic plants for the purpose of edible vaccine.
The antibody response generated by protective antigen expressed in nuclear transgenic tomato plants may be further improved by increasing the expression levels of PA. To meet this objective we employed chloroplast transformation technology and were able to successfully express PA in chloroplasts. The yields obtained in transplastomic samples were uniform and multifold higher as reflected in ELISA and immunoblot results. The expression of anthrax protective antigen in a contained manner has helped in the alleviation of the probable hazards associated with the dispersal of this gene in the environment.
Our study has established the feasibility of expressing PA in a palatable crop that can generate neutralizing antibodies. Chloroplast technology has taken care of the problems that we encountered with nuclear transgenic crops. The high level expression of PA in a palatable crop will be the most promising way to achieve the objective of developing an edible vaccine against anthrax.
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FOOTNOTES
1 Present address: National Institute Of Immunology, Aruna Asaf Ali Marg, New Delhi, India. 
2 Present address: Dartmouth Medical School, Hanover, NH, USA.
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3215fje;
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