transformation of an edible crop with the paga gene of bacillus anthracis

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©2005 FASEB The FASEB Journal express article 10.1096/fj.04-3215fje. Published online July 19, 2005 Transformation of an edible crop with the pagA gene of Bacillus anthracis Mohd. Azhar Aziz,* Deepa Sikriwal,* ,1 Samer Singh* ,2 Sridhar Jarugula, P. Anand Kumar, and Rakesh Bhatnagar* *Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India; and National Research Center For Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India 1 Current address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. 2 Current address: Dartmouth Medical School, Hanover, New Hampshire, USA. Correspondence: Center for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India. E-mail: [email protected] or [email protected] ABSTRACT Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here. Key words: anthrax • protective antigen • chloroplast • biolistics • vaccine nthrax, primarily a disease of herbivores, is caused by Bacillus anthracis. Spores of B. anthracis infect herbivores and can also cause the disease in humans associated with the handling of contaminated animal products. Recently, it has been regarded as one of the most potent bio-terror agents. Studies carried out by Wein et al. (1) have led us to realize the destructive capabilities of B. anthracis. Vaccination against anthrax has been considered one of the most effective prophylactic measures. However, several studies are being reported that describe adverse reactions associated with currently available vaccine formulations (2–4). A Page 1 of 24 (page number not for citation purposes)

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©2005 FASEB

The FASEB Journal express article 10.1096/fj.04-3215fje. Published online July 19, 2005

Transformation of an edible crop with the pagA gene of Bacillus anthracis Mohd. Azhar Aziz,* Deepa Sikriwal,*,1 Samer Singh*,2 Sridhar Jarugula,† P. Anand Kumar,† and Rakesh Bhatnagar*

*Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India; and †National Research Center For Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India

1Current address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.

2Current address: Dartmouth Medical School, Hanover, New Hampshire, USA.

Correspondence: Center for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India. E-mail: [email protected] or [email protected]

ABSTRACT

Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here.

Key words: anthrax • protective antigen • chloroplast • biolistics • vaccine

nthrax, primarily a disease of herbivores, is caused by Bacillus anthracis. Spores of B. anthracis infect herbivores and can also cause the disease in humans associated with the handling of contaminated animal products. Recently, it has been regarded as one of the

most potent bio-terror agents. Studies carried out by Wein et al. (1) have led us to realize the destructive capabilities of B. anthracis. Vaccination against anthrax has been considered one of the most effective prophylactic measures. However, several studies are being reported that describe adverse reactions associated with currently available vaccine formulations (2–4).

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Protective immunity against infection with B. anthracis is based on an immunologic response to the protective antigen (PA; refs. 5–7). The effectiveness of animal and human vaccines depends on the induction of anti-PA antibodies (8). Several efforts are therefore under way to develop safe and acceptable formulations of PA, of which a plant-based vaccine is one. Plants offer an economical, safe, readily acceptable, and easy-to-scale-up expression system for various biomolecules and augurs well for improving health care conditions in developing nations. Several vaccine antigens have been expressed successfully in plants (9–12). The validation of the concept of generating oral immunogenicity by plant-based, edible vaccines has established the faith in this emerging area of vaccine development (13).

We have earlier cloned and expressed PA gene (pagA) coding for PA in tobacco plants (14). The proof of the concept, demonstrating expression of the biologically active PA molecule, in planta, encouraged other groups to develop a new generation plant-based anthrax vaccine (15, 16). Once the proper expression of PA is established in tobacco, there is a need to look for better alternative crops, which are palatable and can be used by human beings in a much easier way. The choice of a crop that can be used as an inexpensive animal fodder will be an added advantage. Among the family of food crops, the tomato fits best into such criteria. The tomato is therefore considered to be a promising crop that can be used to develop an edible vaccine against anthrax.

In the case of edible vaccines, the requirement of antigen is manifold higher than the requirement by the injectable vaccines (12). This fact becomes an area of concern, when nuclear transgenic crops, plagued by the extreme low-expression levels of foreign genes, are used for that purpose (17). The attainment of a threshold level of foreign gene expression is a necessity, when we look into the problem of generating tolerance as a result of consumption of transgenic plants expressing suboptimal levels of protein. Recent environmental concerns about genetic contamination by transgenic plants have demanded an alternative to nuclear transformation of plants (18, 19). These problems of nuclear transgenic crops have been addressed with the advent of chloroplast transformation technology (20).

The development of a chloroplast transformation system has ushered plant biotechnology into a new phase with exciting possibilities of high-level gene expression (21, 22). The plastid genome of higher plants is an attractive target for engineering, as it provides readily obtainable, high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission (23). Chloroplast transformation typically uses two flanking sequences, which through homologous recombination, insert trangenes into spacer regions between functional genes of the chloroplast genome, thus targeting the transgene to a known location (24). In higher plants, plastid transformation is obtained routinely in tobacco.

This report describes the expression of PA in nuclear transgenic tomato plants, which may serve as a source of an edible vaccine against anthrax. The total soluble protein (TSP) extracted from transgenic tomato leaves was used to study the generation of PA-specific antibodies in mouse animal models. The PA expressed in transgenic tomato plants was able to generate a population of neutralizing antibodies, although at low titer values. These titer values can be improved by increasing the expression levels of PA. For this reason, chloroplast transformation technology was used, and increased expression levels of PA in an experimental crop, tobacco, were recorded.

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MATERIALS AND METHODS

Bacterial strains and culture conditions

Two strains of Escherichia coli, DH5α and XL1 Blue, were used for routine maintenance and propagation of plasmids (Table 1). They were grown in Luria Bertani broth media supplemented with ampicillin (100 mg/liter), kanamycin (50 mg/liter), or spectinomycin (50 mg/liter) as necessary. A. tumefaciens strain LBA 4404 (27) was grown routinely in yeast extract mannitol medium (14) supplemented with kanamycin (50 mg/liter) and rifampicin (10 mg/liter) when required. Antibiotics and other media components were procured from USB Corp. (Cleveland, OH, USA). Unless otherwise stated, all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Generation of transgenic tomato plants

The cotyledonary leaves from 10- to 12-day-old tomato seedlings were used for leaf-disc transformation as described elsewhere (28) with some modifications and preliminary standardizations. Briefly, the infection time of agrobacterium and the use of silver nitrate were optimized for the transformation protocol. The vector used for tomato transformation was constructed by the methodology described earlier (14). Briefly, pagA was cloned in a binary vector, pBINAR, carrying a constitutive CaMV 35 S promoter and a kanamycin selection gene, neomycin phosphotransferase (nptII). The transformed leaf discs were subjected to several rounds of kanamycin (100 mg/liter) selection. Growth media and its components were from Hi Media Laboratories (India). Initially, phytohormones—zeatin (0.1 mg/liter), ∝-naphthalene acetic acid (0.1 mg/liter), and benzyl aminopurine (1 mg/liter)—were used with Murashige-Skoog (MS) media to induce callus stage. Later, transferring the shoots to MS media without hormones induced rooting. Finally, plants were potted in a vermiculite mixture under controlled conditions of temperature, photoperiod, and humidity. Wild-type, nontransformed plants were grown alongside under the same conditions and were used as a negative control in all experimental analyses.

Cloning pagA in a chloroplast vector

pagA was cloned in a chloroplast vector pLD-Ctv (~5.9 kb), a kind gift from Professor Henry Daniell (University of Central Florida, Orlando, FL, USA). pagA was polymerase chain reaction (PCR)-amplified from pMW plasmid using specific primers, CPI (5′ AAG GAA AAA AGA TAT CAG GAG GTT TAT ATG GAA GTT AAA CAG GAG AAC CGG 3′) and CPII (5′ AAA AGG AAA AGC GGC CGC TTA TCC TAT CTC ATA GCC TTT TTT AGA AAAG 3′). Forward primer was designed to introduce ribosomal binding site (GGAGG), spacer (TTTAT), initiation codon (AUG), and a restriction site for the EcoRV enzyme. Reverse primer was designed to introduce the NotI site at the 3′ end of PA gene. The resulting clone, pLD-PA gene, was subjected to restriction analysis followed by automated sequencing using an ABI 377 prism automated sequencer (Applied Biosystems International, Foster City, CA). The sequence of engineered pagA is submitted to GenBank, Accession Number AY 700758.

pLD-PAG was transformed in competent XL1 Blue E. coli cells for checking the expression of PA on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed

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by immunoblot detection by polyclonal antibodies raised against purified recombinant PA (rPA). Standard molecular biology methods were used for cloning and analysis of pLD-PAG (29).

Generation of transplastomic samples

Biolistics was used to transform tobacco chloroplast by standard bombardment protocol (30). The explant used for particle bombardments was a fully grown tobacco leaf placed on an osmoticum 24 h prior to bombardment. Different compositions of osmoticum were used for testing the bombardment efficiency. After the bombardment, the leaves were kept in the dark for 48 h, followed by cutting them into small pieces (~5×5 mm) and kept on selection medium [MS salts and phytohormones along with spectinomycin (500 mg/liter)]. Two rounds of selection were carried out at an interval of 2–3 months. Tobacco leaves bombarded with pLD-Ctv (only vector DNA) were considered as controls for all molecular analysis experiments. The transformed tobacco samples with the pagA gene integrated in the chloroplast genome were “transplastomics”. Accessories and components of Gene gun PDS-1000/He was purchased from its sole distributor, Bio-Rad (Hercules, CA, USA).

Transgene analysis of putative transformants

Putative transgenic tomato plants were subjected to PCR analysis and Southern hybridization to ascertain the pagA integration in the nuclear genome. Genomic DNA (gDNA) (10–50 ng) extracted from the samples using Qiagen kits was used as a template for PCR analysis of tomato samples as described earlier (14). The PCR-amplified products were digested with the EcoRI enzyme to further authenticate their composition.

To investigate the integration of pagA in the nuclear genome of the tomato plants, Southern hybridization was carried out using phosphate buffer protocol. gDNA (12 µg) was completely digested with EcoRI and resolved on a 0.6% agarose gel followed by capillary transfer blotting onto a charged nitrocellulose membrane (29). Plasmid pBIN-PAG was taken as positive control, whereas gDNA from nontransformed tomato plants served as a negative control. The membranes for DNA gel blotting were purchased from Amersham Biosciences (Piscataway, NY, USA). The probe was prepared by random primer labeling of a 1.5-kb fragment obtained after digesting pBIN-PAG (14) with EcoRI.

Transplastomic samples of tobacco obtained after application of biolistics were analyzed for the presence of pagA using the PCR technique. Terminal primers were used to amplify full-length pagA from gDNA using the same protocol as in ref. (14) with some modifications.

Analysis of TSP

Extraction of TSP and immunoblot detection

The transplastomic samples of tobacco and the leaves of putative transgenic tomato plants were crushed separately in liquid nitrogen, and the resultant powder was suspended in extraction buffer [20 mM HEPES buffer+5% (v/v) glycerol] supplemented with protease inhibitor cocktail. Sample (100 mg) was suspended in 100 µl extraction buffer and centrifuged at 17,000 rpm to remove insoluble material. Supernatant was stored at 4°C for further analysis. All the steps of protein extraction were carried out at 4°C. These protein samples were subjected to SDS-PAGE analysis followed by immunoblot detection as described earlier (14). Briefly, primary polyclonal

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antibodies against PA were used at a dilution of 1:5000 for transplastomic samples, whereas TSP from transgenic tomato plants was probed at a dilution of 1:200. Horseradish peroxidase (HRP) enzyme-conjugated secondary antibodies were used to generate colored reaction product using 3-3′ diaminobenzidine as a substrate.

Quantification of PA

The expression of PA in TSP extracted from transplastomic tobacco samples was quantitated by using enzyme-linked immunosorbent assay (ELISA). The protein samples were suspended in phosphate-buffered saline and coated on a 96-well, round-bottomed ELISA plate (BD Transduction Labs, Franklin Lakes, NJ, USA). Purified PA (1 µg), 1–16 µg transplastomic protein samples, and 15 µg control sample (from untransformed plants) were coated in each well. After overnight coating with the proteins, the residual spaces in the well were blocked by 2% bovine serum albumin. Polyclonal antibodies against rPA were added to these wells. A gradient of antibody dilutions increasing by a factor of two was maintained across the plate. This was followed by the addition of HRP-conjugated anti-rabbit secondary antibodies. 3,3′,5,5′ Tetramethylbenzidine (TMB) substrate was added after 2 h incubation, followed by reading the absorbance at 630 nm using a Bio-Rad ELISA plate reader. The absorbance recorded in the wells containing protein extracted from transplastomic samples was compared with that of a standard plot of purified PA to obtain the percentage expression levels of PA in transplastomic samples. The absorbance values fitting to the linear region of the plot were taken into consideration. All the reactions were done in triplicate.

In vitro functional studies of plant-expressed PA

An in vitro assessment of cytotoxic activity of a functional PA molecule was carried out in the presence of lethal factor (LF)—a component of the tripartite anthrax toxin (31). Different samples of the protein extracted from transplastomic samples of tobacco and transgenic tomato leaves were mixed separately with LF (at a final concentration of 500 ng/ml) and added onto a macrophage-like cell line, RAW 264.7, maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS). The protocol followed for the assay was the same as described in ref. (14). All the experiments were carried out in triplicate. Cell culture plasticware was obtained from Corning (Corning, NY, USA). FCS was from Biological Industries (Israel).

In vivo studies on mouse models

Groups of five Balb/c inbred mice (National Institute of Nutrition, Hyderabad, India), 3–4 weeks old, weighing 18–20 gm, were used for immunization studies. All the experiments were conducted strictly in accordance with Indian Animal Ethics Committee regulations. Concentrated TSP extracted from tomato leaves was mixed with complete Freund’s adjuvant (for first dose) and incomplete Freund’s adjuvant (for subsequent doses) in a ratio of 1:1 to prepare the injection samples. Adjuvants were procured from Difco Laboratories (Detroit, MI, USA). Three booster doses of protein samples were administered to the animal groups on days 14, 28, and 60 after the primary dose (Day 0). TSP (300 µg) was administered to groups I (transgenic tomato) and II (wild-type tomato), and Group III animals received 30 µg rPA. Blood was collected 1 day prior to the date of injection. Serum extracted from blood samples was analyzed for the presence of PA-specific antibodies by using purified rPA-based, direct ELISA. End-point titers were calculated using the reciprocal of the highest serum dilutions that had an absorbance ≥0.1 optical

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density units above background. Neutralization assay with these antisera were carried out as described earlier (32).

RESULTS

Transgenic tomato plants with pagA incorporated in its nuclear genome

Transgenic tomato plants were generated using an agrobacterium-mediated leaf-disc transformation method. Slight modifications in regeneration protocol were helpful in getting a good number of transformants. There were no phenotypic aberrations observed in transgenic tomato plants, which were more sensitive to fluctuations in temperatures and humidity, and the wild-type plants were able to withstand such conditions. The integration of the pagA gene in the nuclear genome was studied using PCR-based analysis of tomato gDNA, followed by Southern hybridization. An amplification of 1.5 kb was obtained by using primers, PAI (5′ GGA TTG GAT TTC AAG TTG TAC TGG ACC 3′) and PAII (5′ CTT AAC TAC TGA CTC ATC CGC CCC AAC 3′), targeted at locations +384 and +1840 of pagA (Fig. 1A). This amplified product was digested by an EcoRI enzyme, which yielded two fragments of 1.0 kb and 0.5 kb (Fig. 1C), which is in conformity with the presence of an EcoRI site in this region. nptII-specific primers were able to amplify a 0.7-kb fragment in selected plants (Fig. 1B). There was no amplification observed with these two sets of primers, in nontransformed, wild-type tomato plants.

Southern hybridization results showing signals from a pagA-specific probe, corroborated the above results. A 1.5-kb fragment encompassing a part of the CaMV 35S promoter and pagA was able to hybridize with gDNA of selected transgenic plants (Fig. 2). There were no such bands visible in nontransformed, control samples, even after longer periods of hybridization and exposure.

Expression of PA in transgenic tomato plants

The fidelity of PA protein expression in transgenic tomato plants was confirmed by the detection of a single protein of 83 kDa by anti-PA polyclonal antibodies at a maximum dilution of 1:200. The intensity of the signals from protein extracts varied from one plant to another. However, there was no significant difference in expression levels of PA with respect to the age of the leaf samples from the same plant (data not shown). The absence of any band in a lane carrying TSP extracted from an untransformed plant ruled out the possibility of cross-reaction of antibodies with other plant proteins. The TSP from transgenic plants (72 µg) was able to kill macrophage cells in the cytotoxicity assay (Fig. 3). The macrophage cells receiving TSP along with LF [a component of lethal toxin (LeTx)=LF+PA] show a percentage killing in the range of 20–77%. There was 97–100% survival of cells in an environment containing only TSP (devoid of LF), only buffer, or only LF.

Animal experiments

Generation of PA-specific antibodies

The PA expressed in nuclear transgenic tomato plants was able to generate an antibody-mediated immune response. The presence of PA-specific antibodies in the sera of immunized animals was detected and quantitated by ELISA. The antibody titers obtained in the test sample were of the

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order of 1600, as measured before injecting a second booster dose (Table 2). This level of antibody titer was maintained until the last record, which was done at Day 59. rPA used as a positive control was able to generate antibody titers of the order of ≥50,000 under similar conditions, whereas TSP from nontransformed plants was not able to record any appreciable absorbance after addition of the TMB substrate.

Neutralization of LeTx action

Neutralization (in vitro protection) assays show the prevention of killing of macrophage cells by LeTx in the presence of sera isolated from the immunized animals (Group I). Sera from these animals caused a neutralizing effect on RAW264.7, a macrophage-like cell line, at a maximum dilution of 1:100. Sera from Group II animals showed no neutralization effect, whereas Group III animals were able to show a strong neutralizing effect at this dilution (Fig. 4).

Construction of plastid transformation construct carrying PAG: pLD-PAG

The 2.2-kb pagA gene was inserted into the universal chloroplast expression vector to form the final E. coli and tobacco shuttle vector, pLD-PAG. This vector has a universal plastid rRNA (Prrn) operon promoter, an antibiotic selection marker gene (aadA) coding for resistance against aminoglycosides, and homologous recombination sites of trnI and trnA genes for the insertion of transgene in chloroplast genome. The presence of the TpsbA 3′ untranslated region is the stabilizing factor for the transcripts. An illustration of the chloroplast vector after the insertion of transgene is depicted in Fig. 5A.

Unique restriction enzyme sites for EcoRV and NotI were used to clone the PCR-amplified pagA gene. The restriction analysis results with NotI and EcoRI enzymes confirmed the presence of pagA in the pLD-PAG construct. A linearized band of ~8.1 kb was obtained with NotI, whereas EcoRI digestion resulted in a fallout of ~1.0 kb (Fig. 5B). Further, automated sequencing corroborated the results and confirmed the presence of upstream elements added to the pagA using PCR.

Bacterial colonies harboring pLD-PAG were able to grow on spectinomycin (50 mg/liter) and express PA as deduced from SDS-PAGE analysis followed by immunoblot detection. A single band of 83 kDa in a Western blot of pLD-PAG carrying bacterial colonies was observed, whereas there was no such band in colonies carrying vector alone or untransformed XL1 Blue cells (data not shown).

Generation of transplastomics

The osmoticum composition played a crucial role in bombardment experiments. A combination of MS salts, 2% sucrose, 0.5 M sorbitol, and 0.5 M mannitol, along with agar was able to give the best results. Bombarded leaf samples were subjected to spectinomycin selection, and the selected transplastomic samples were taken for transgene analysis. Each transformed explant grew into a callus, whereas control leaf explants turned white without forming a callus. gDNA extracted from these samples was subjected to PCR analysis using primers corresponding to 5′ and 3′ termini of 2.2 kb pagA. All the leaf explants bombarded with pLD-PAG, which were able to grow on spectinomycin (500 mg/liter), exhibited amplification of appropriate size, whereas no amplification was observed in control plants (Fig. 6).

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Analysis of transplastomic TSP

TSP extracted from selected transplastomic samples was able to show distinct bands of PA in Coomassie-stained SDS-PAGE analysis as compared with a control sample (Fig. 7). Immunoblot analysis of these proteins detected an 83-kDa protein molecule, when probed with polyclonal anti-PA antibodies (Fig. 7). The amount of PA in TSP, as measured by direct ELISA using rPA as a reference, gave an estimate of 8% of PA protein in transplastomic samples (Fig. 8). There was no significant variation in the expression levels of PA among the transplastomic samples.

These samples, when added to the media of macrophage cells along with LF, displayed a cytotoxic effect, as inferred from an in vitro macrophage killing assay. Quantitative estimation of this effect, by adding 30 µg TSP to the environment of the macrophage-like cell line, showed 97% killing (Fig. 9). No lethal effect was observed when cells were exposed to TSP extracted from control plants.

DISCUSSION

This study describes two important achievements toward the ultimate goal of generating an edible vaccine against anthrax. First, the successful expression of functional PA in the tomato was achieved, and second, the tobacco chloroplasts were transformed with pagA to obtain multifold expression levels of functionally active PA.

The prognosis of anthrax infection is worst in the case of pulmonary and gastrointestinal anthrax (33). An efficient way to protect an individual would be to counter the infection at the first site of defense, i.e., mucosal lining. Oral vaccines will thus be favored over the conventional injectable vaccines. Moreover, oral vaccines have been reported to induce a dual response involving T and B cells (34), which is needed to generate immunity against anthrax (32). An edible vaccine will be able to address the concern of anthrax disease among animals as well as humans. Our initial studies (14) were followed by the expression of anthrax immunogen in a palatable crop, spinach (15). This method of generating edible vaccine is labor-intensive and involves the need of technical experts, which hampers the idea of producing a cost-effective vaccine against anthrax. However, the need to express PA in a palatable crop remained. Other reports of pagA expression (16) are encouraging in terms of good protein yields, but the studies need a further validation in terms of immunogenic characterization of the plant-expressed antigen.

Among the family of food crops, the tomato represents the best candidate when considering the formulation of a readily acceptable vaccination regimen. In this report, we expressed PA in tomato plants using pBIN-PAG as the construct carrying the pagA gene under the influence of a strong, constitutive promoter (14). Regeneration of tomato plants following agrobacterium-mediated leaf-disc transformation was more tedious and cumbersome, owing to their excessively prone nature to contamination. Silver nitrate was used at a concentration of 20 µM to counter this problem effectively (25). We were able to regenerate tomato plants successfully and transfer them to the pot stage. The selected plants were subjected to molecular analysis, and the PCR results followed by Southern hybridization confirmed the integration of pagA in a nuclear genome of tomato plants. The presence of PA in TSP was confirmed by immunoblot results, where an 83-kDa band was lit up when probed with polyclonal PA antibodies. The variability in expression levels of PA in different plants is in accordance with earlier reports (35), but unlike other reports (36), our results show no significant variance in expression levels of PA with

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respect to the age of the leaf. The functional efficacy of the PA expressed in nuclear transgenic tomato plants was confirmed using an in vitro macrophage killing assay (37). The functional PA expressed in nuclear transgenic tomato plants was checked for its ability to generate an antibody response in mouse models. ELISA results confirmed the ability of plant-expressed PA to generate antibody titers, although at a low level. The in vitro protection assay results indicated the presence of a neutralizing antibody population. These results confirm the ability of plant-expressed PA to generate an antibody-mediated immune response. However, the concern of low antibody titers, which are mainly attributed to the extremely low expression level observed in nuclear transgenic crops, remains (38, 39). As generation of humoral response has been correlated with the protective efficacy of the anthrax vaccine (32), there is a need to improve the antibody titers in case of transgenic plants expressing PA. There can be several factors that may contribute toward enhancing the antibody titers (39). We focused our efforts on increasing the expression levels of PA in plants so that more of an amount of antigen can be administered to the vaccinees. This is more in consonance with our objective to generate an edible vaccines against anthrax. The idea of edible vaccine demands maximal antigen production and a consistent expression profile to design a particular regimen for the vaccinees. Low expression levels may result in an inability to generate a protective immune response and create tolerance in an individual (40). Another dimension of creating nuclear transgenic plants is in an environmental perspective. There may be unfound fears of spreading pagA gene in the environment leading to unknown effects. This hurdle of low and inconsistent expression and scare of genetic contamination motivated us into the area of chloroplast engineering, and we attempted generating transplastomic tobacco plants.

With the advent of technologies for transforming the genetic composition of chloroplasts, a new, attractive target for genetic engineering has become available (23). High protein-expression levels (22, 41), absence of epigenetic effects (gene silencing, position effects), and easy transgene stacking in operons make the use of plastid genome engineering attractive (24). From the bio-safety perspective, the technology significantly increases transgene containment, as plastids are maternally inherited in most crops and are not transmitted by pollen (24, 42).

A chloroplast transformation vector pLD-PAG was constructed to express pagA in a chloroplast-specific manner. The pagA with additional upstream elements (Shine-Dalgarno sequence, spacer, and initiation codon) was combined with aadA to form an operon. This vector has been used successfully in tobacco and is expected to work in other crops as well, owing to the use of highly conserved, homologous regions (22). The Prrn operon promoter in higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a σ70-type promoter with conserved –10 and –35 core promoter elements (43). Given the conservation of the E. coli and PEP transcription machineries (44), we envisaged checking the expression of PAG in E. coli prior to starting the actual chloroplast-bombarding experiments. Immunoblot results confirmed the expression of pagA in E. coli.

The addition of an osmoticum (i.e., a supplemental agent increasing osmolarity) to the bombardment medium can dramatically increase the rates of transformation using biolistics. Elevated osmoticum concentration may work by protecting the cells from leakage and bursting (low turgor pressure) and may also improve particle penetration itself (30). It was clearly evident that medium containing mannitol and sorbitol made the best combination for carrying out bombardments on tobacco leaves.

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The molecular analysis of the callus obtained on the selection medium confirmed the successful integration and subsequent expression of PAG in the transplastomic samples. The targeted gene integration event is a characteristic feature of chloroplast transformation and obviated the need to screen a large number of plants as required in nuclear transgenics (23). Thus, a few selected PCR-positive transplastomic lines were followed for further analysis. A higher expression level of PA was obtained, as evident by immunoblot analysis at a higher dilution of antibodies (1:5000). The higher levels of PA in TSP were validated by results of direct ELISA. A comparison of absorbance obtained with TSP and rPA protein gave an estimate of an 8% PA yield. The higher yield of the PA in TSP can be attributed to several reasons of which the presence of a high ploidy level is considered to be most plausible (21). This ploidy level is of the order of 10,000 gene copies per cell. Expression of PA in the form of native bacterial coding region was favored, probably because of the stability of the mRNA transcribed from pagA. Although plants are eukaryotes, their plastids share some feature of prokaryote machinery for mRNA turnover (45). An efficient translation of the bacterial A/T-rich PA (69%) coding region reflects the plastid genome codon bias, which is also relatively A/T-rich (46). A significant improvement in the protein yields is encouraging to embark on the task of transforming edible crops to generate an edible vaccine against anthrax.

ACKNOWLEDGMENTS

This work was partially supported by the National Agricultural Technology Project. M. A. A. was a recipient of a senior research fellowship from the Council of Scientific and Industrial Research. Chloroplast vector pLD-PAG was a kind gift of Prof. H. Daniell at the University of Central Florida. The help extended by Ms. Meenakshi Sharma in preparing the manuscript is duly acknowledged. The expertise of Dr. Mohd. Ayub Qadri (National Institute of Immunology, New Delhi, India) in designing and interpreting animal experiments is gratefully acknowledged.

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Received November 21, 2004; accepted June 8, 2005.

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Table 1 Plasmids and strains used in this study Strains or plasmid Description Reference/source 1. Bacterial strains Escherichia coli; DH5α and XLI Blue Host strains for cloning and Lab stock maintenance of plasmids Agrobacterium tumifaciens Host strain for leaf disc transformation (25) LBA 4404 2. Plant strains Nicotiana tabacum Tobacco for generating transplastomics Pusa, Indian Agricultural var. benthamiana Research Institute (IARI) Lycopersicon esculentum var. Pusa Ruby Tomato for leaf disc transformation Pusa, IARI 3. Plasmids pBINAR Binary vector P. A. Kumar pBIN-PAG Binary pagA construct (14) pLD-Ctv Chloroplast expression vector Daniell and co-workers (22) pLD-PAG Chloroplast pagA construct This study pMW Vector containing pagA open- reading frame (26)

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Table 2 Generation of PA-specific antibody response by plant-expressed protective antigen Group of animals Mean antibody titres I 1600 II Nil III >50,000 Test group of animals receiving TSP from transgenic plants (I), untransformed tomato plants (II), and recombinant PA (III).

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Fig. 1

Figure 1. PCR analysis of putative transgenic tomato plants. Genomic DNA extracted from the leaves was used as template. A) PCR amplification of an internal pagA region amplified by primers directed at +384 and +1840 locations. An amplification of ~1.5 kb was obtained. T1–T5 show amplifications from five representative transgenic plant samples. B) PCR amplification of nptII using specific gene primers. An amplification of ~0.7 kb was observed in five representative samples (T1–T5), whereas untransformed plants (U) were not able to show any such amplification. C) Restriction analysis of the fragment amplified from pagA. The amplified products were purified and digested with EcoRI enzyme. The appearance of ~1.0 kb and 0.5 kb bands is in accordance with the presence of the enzyme site in this region. T1–T3, Representative transgenic plant samples; M, 1 kb gene ladder from MBI Fermentas (Lithuania).

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Fig. 2

Figure 2. Integration of pagA into nuclear genome of transgenic tomato plants. Southern hybridization of genomic DNA extracted from representative plant samples was carried out. Genomic DNA (12 µg) was digested with EcoRI enzyme, resolved on a 0.6% agarose gel, and blotted on a charged membrane. The membrane was probed with a 32P-labeled, pagA-containing fragment. PC, Plasmid DNA, pBIN-PAG; T1–T3, representative transgenic samples; U, untransformed tomato plant sample.

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Fig. 3

Figure 3. Biological activity of PA expressed in nuclear transgenic tomato plants. Equal amounts of TSPs (72 µg) isolated from transgenic plants (T1–T5) and untransformed plants (U) were added to macrophage cells along with LF (500 ng/ml). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye was added after 16 h incubation at 37°C. Quantitative measurements were done by recording the absorbance at 540 nm. Percentage killing was calculated by comparison with 100% killing shown by 1 µg rPA with a similar amount of LF.

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Fig. 4

Figure 4. Generation of neutralizing antibodies by plant-expressed PA. The antisera generated in immunized animals were incubated with functional PA and added along with LF onto macrophage cells. The killing of cells was measured by MTT dye assay. Three dilutions of antisera were tested. Antisera from animals immunized with (I) TSP extracted from nuclear transgenic tomato plants, (II) TSP extracted from untransformed tomato plants, and (III) rPA were used to study the neutralizing effect.

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Fig. 5

Figure 5. Construction of chloroplast transformation vector. A) Schematic representation of the chloroplast vector after cloning of modified pagA in EcoRV and NotI enzyme sites. B) Restriction analysis of the chloroplast transformation vector, pLD-PAG. M, 1 kb gene ladder; 1, pLD-Ctv digested with EcoRI; 2, pLD-PAG digested with EcoRI; 3, pLD-PAG digested with EcoRV; 4, pLD-PAG digested with NotI.

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Fig. 6

Figure 6. PCR analysis of pagA in transplastomic samples. Specific amplification of pagA in transplastomic samples was carried out using CPI and CPII end primers. An amplification product of ~2.2 kb was obtained in representative transplastomic samples (C1–C3), whereas there was no such amplification in an untransformed transplastomic sample (U). M, 1 kb gene ladder.

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Fig. 7

Figure 7. Coomasie-stained SDS-PAGE profile of PA expressed in a transplastomic sample. The appearance of a distinct band in the transplastomic sample is indicative of the expression of PA in good amounts. The Western blot results (lower panel) confirm the identity of the band as PA. There was no such band visible in untransformed plants.

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Fig. 8

Figure 8. Quantitative estimation of PA expressed in transplastomic samples. A direct ELISA was carried out using TSP extracted from three representative samples (C1–C3). The absorbance obtained at different dilutions was compared with a standard plot of PA. The amount of PA detected in transplastomic samples was consistently recorded at ~8% of total soluble protein. There was no recordable absorbance in TSP extracted from untransformed (U) samples.

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Fig. 9

Figure 9. Functional efficacy of PA expressed in transplastomic samples. A MTT dye-based cytotoxicity assay on macrophage-like cell line RAW 264.7 was carried out. The biological activity of the PA in transplastomic samples (C1–C3) was found at par with the rPA. No cytotoxicity was observed in untransformed samples.

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